Novel immunogenic peptides

ABSTRACT

The invention relates to isolated immunogenic peptides comprising a MHC class II T cell epitope, and immediately adjacent or separated from said epitope a H-X(0,2)-C-X(2)-[CST] or [CST]-X(2)-C-X(0,2)-H redox motif.

FIELD OF THE INVENTION

The present invention relates to immunogenic peptides. The peptides areused in in vitro and in vivo systems to generate antigen specificcytolytic CD4+ T cells. The peptides and cells obtained by thesepeptides are used as pharmaceutically active peptides for a variety ofdisorders including auto immune diseases such as multiple sclerosis.

BACKGROUND OF THE INVENTION

WO2008/017517 discloses a novel class of peptides which comprise an MHCclass II T cell epitope of an antigen and a redox motif sequence.

Redox motif sequences have been reviewed in Fomenko et al. (2003)Biochemistry 42, 11214-11225. The different alternatives of the redoxmotif sequence are C(X)2C [SEQ ID NO:71], C(X)2S [SEQ ID NO:72], C(X)2T[SEQ ID NO:73], S(X)2C [SEQ ID NO:74], and T(X)2C [SEQ ID NO:75]. Otherprior art on redox motif sequences comments on the relevance of aHistidine within the redox motif sequence [Kortemme et al. (1996)Biochemistry 35, 14503-14511].

WO2008/017517 explains that the combination of a T cell epitope and aredox motif sequence in each other's proximity within a peptide providesproperties which have not been recognised before. Namely, such peptideshave the capacity to elicit a population of CD4+ cytolytic T cells whichkill specifically the antigen presenting cells which present the antigencomprising the T cell epitope which is present in the peptide.

Consequently these peptides can be used to block an immune response at avery early stage, i.e. at the level of antigen presentation.WO2008/017517 demonstrates the medical use of these peptides in thetreatment and prevention of allergies and immune disorders. The conceptof the invention has been later published in Carlier et al. (2012) Plosone 7, 10 e45366. Further patent applications demonstrated that suchpeptides can be used in other medical applications wherein immuneresponses are to be avoided, such as the treatment of tumours,rejections of transplants, immune responses against soluble allofactors,immune responses against viral proteins encoded by the backbone of viralvectors.

The above publications discuss the type of redox motif sequence and thespacing between redox motif and T cell epitope sequence. Furtherdeterminants in the peptides which may provide improved properties tothe peptides have not been reported.

SUMMARY OF THE INVENTION

The different alternatives of the 4 amino acid redox motif sequence asmentioned in the introduction can also written as [CST]-X(2)-C [SEQ IDNO:76] or C-X(2)-[CST][SEQ ID NO:77]. The present invention reveals thatthe presence of an additional Histidine amino acid immediately adjacentoutside the motif (N terminal of the motif (position −1) or C-terminalof the motif (position +5)) increases the stability of the redox motif.Thus, the present invention relates to modified redox motifs withgeneral structure or H-C-X(2)-[CST] [SEQ ID NO:78] or [CST]-X(2)-C-H[SEQ ID NO: 79].

With this improved stability the specific reducing activity of thepeptide increases, such that for example less peptide can be used or thenumber of injections is reduced, compared to a peptide wherein theadditional Histidine is not present.

A first aspect relates to isolated immunogenic peptidea of between 13and 100 amino acids comprising a MHC class II T cell epitope of anantigen, and immediately adjacent or separated by at most 7 amino acidsfrom said epitope a H-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78], [SEQ IDNO:90] or [SEQ ID NO:91]) or a [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79],[SEQ ID NO:92] or [SEQ ID NO:93]) redox motif sequence for use as amedicament.

In certain embodiment said antigen does not contain in its sequence saidmotif within a distance of 10 amino acids of said epitope, or even doesnot contain in its sequence said motif.

in specific embodiments the motif is H-X-C-X(2)-[CST] [SEQ ID NO:90] or[CST]-X(2)-C-X-H [SEQ ID NO:92] redox motif sequence, or isH-C-X(2)-[CST] [SEQ ID NO:78] or [CST]-X(2)-C-H [SEQ ID NO:79] redoxmotif sequence.

In other embodiments the motif is H-X(0,2)-C-X(2)-C([SEQ ID NO:80], [SEQID NO:96] or [SEQ ID NO:97]), or C-X(2)-C-X(0,2)-H ([SEQ ID NO:83], [SEQID NO:94] or [SEQ ID NO:95]).

In yet other embodiments the motif is H-C-X(2)-C [SEQ ID NO:80] orC-X(2)-C-H [SEQ ID NO: 83].

In specific embodiments, the peptides have a length of between 13 and 75amino acids, between 13 and 50 amino acids, or between 13 and 30 aminoacids.

The MHC class II T cell epitope, can separated from said motif by asequence of at most 4 amino acids, or by a sequence of 2 amino acids.

In specific embodiments, wherein X within the redox motif is Gly or Pro,or X within the redox motif is not Cys.

In other specific embodiment, X outside the redox motif is not Cys, Seror Thr.

The peptides can be used in the prevention or treatment of multiplesclerosis (MS), whereby the antigen is an auto-antigen involved inmultiple sclerosis, such as MOG. Specific embodiments of a peptide forMS comprise the epitope sequence VVHLYRNGK [SEQ ID NO:3], such asHCPYCSRVVHLYRNGKD [SEQ ID NO:1], HxCPYCSRVVHLYRNGKD [SEQ ID NO: 115], orHxxCPYCSRVVHLYRNGKD [SEQ ID NO: 116].

The peptides can be used in the prevention or treatment of diabetes,wherein the antigen is for example proinsulin.

Another aspect relates to isolated immunogenic peptides of between 13and 100 amino acids comprising a MHC class II T cell epitope of anantigen, and immediately adjacent or separated by at most 7 amino acidsfrom said epitope a H-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78] or [SEQ IDNO:90] or [SEQ ID NO:91]) or [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79], [SEQID NO:92] or [SEQ ID NO:93] redox motif sequence, with the proviso thatsaid antigen does not contain in its sequence said motif within adistance of 10 amino acids of said epitope.

In certain embodiment the antigen does not contain in its sequence saidmotif. Specific embodiments of motifs are H-X-C-X(2)-[CST] [SEQ IDNO:90], [CST]-X(2)-C-X-H [SEQ ID NO:92], H-C-X(2)-[CST] [SEQ ID NO:78]or [CST]-X(2)-C-H [SEQ ID NO:79], X(0,2)-C-X(2)-C([SEQ ID NO: 80], [SEQID NO:96] [SEQ ID NO:97]), C-X(2)-C-X(0,2)-H ([SEQ ID NO:83], [SEQ IDNO:94] [SEQ ID NO:95]) H-C-X(2)-C [SEQ ID NO:80] or C-X(2)-C-H [SEQ IDNO:83].

In specific embodiments of peptides, if said motif isH-X(0,2)-C-X(2)-[CST] [SEQ ID NO:78, 90 or 91], the motif is located Nterminally from the T cell epitope within the peptide, and wherein, ifsaid motif is [CST]-X(2)-C-X(0,2)-H [SEQ ID NO:79, 92 or 93], the motifis located C terminally from the T cell epitope.

The motif can located N terminally from the T cell epitope. The peptidescan have a length of between 13 and 75 amino acids, of between 13 and 50amino acids, of between 13 and 30 amino acids.

In specific embodiments, the MHC class II T cell epitope, is separatedfrom said motif by a sequence of at most 4 amino acids or is separatedfrom said motif by sequence of 2 amino acids.

In specific embodiments X within the redox motif is Gly or Pro, or Xwithin the redox motif is not Cys.

In specific embodiments X outside the redox motif is not Cys, Ser orThr.

Particular peptides are from the auto-antigen is MOG or proinsulin.

Particular peptides comprise the epitope sequence VVHLYRNGK [SEQ IDNO:3], such as HCPYCSRVVHLYRNGKD [SEQ ID NO:1], HxCPYCSRVVHLYRNGKD [SEQID NO:115], or HxxCPYCSRVVHLYRNGKD [SEQ ID NO:116].

Another aspect are methods of treatment or prevention comprising thestep of administering an effective amount of an immunogenic peptide ofbetween 13 and 100 amino acids comprising an MHC class II T cell epitopeof an antigen, and immediately adjacent or separated by at most 7 aminoacids from said epitope a H-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78], [SEQ IDNO:90] or [SEQ ID NO:91]) or a [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79],[SEQ ID NO:92] or [SEQ ID NO:93]) redox motif sequence.

Another aspect of the invention relates to in vitro use of a describedabove for the generation of antigen specific CD4+ cytolytic T cells.

Another aspect relates to a method for obtaining a population CD4+ Tcells which are cytolytic against cells antigen, the method comprisingthe steps of: providing peripheral blood cells; contacting said cells invitro with an immunogenic peptide of between 13 and 100 amino acidscomprising an MHC class II T cell epitope of an antigen, and immediatelyadjacent or separated by at most 7 amino acids from said epitope aH-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78], [SEQ ID NO:90] or [SEQ ID NO:91])or a [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79], [SEQ ID NO:92] or [SEQ IDNO:93]) redox motif sequence; and expanding said cells in the presenceof IL-2.

Another aspect relates to a population of cells obtainable by the abovemethod of for use as a medicament.

Another aspect relates to methods of treatment and prevention comprisingthe step of administering an effective amount of cells as describedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Response of naive human CD4+ T cell lines towards peptides witha T cell epitope of MOG and a redox motif without (right bars) [SEQ IDNO:7] and with additional Histidine (left bars). [SEQ ID NO:1]

DETAILED DESCRIPTION Definitions

The term “peptide” as used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can comprise non-amino acid structures.Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino-acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification. The term “antigen”as used herein refers to a structure of a macromolecule, typicallyprotein (with or without polysaccharides) or made of proteic compositioncomprising one or more hapten (s) and comprising T cell epitopes. Theterm “antigenic protein” as used herein refers to a protein comprisingone or more T cell epitopes. An auto-antigen or auto-antigenic proteinas used herein refers to a human or animal protein present in the body,which elicits an immune response within the same human or animal body.

The term “food or pharmaceutical antigenic protein” refers to anantigenic protein naturally present in a food or pharmaceutical product,such as in a vaccine. The term “epitope” refers to one or severalportions (which may define a conformational epitope) of an antigenicprotein which is/are specifically recognised and bound by an antibody ora portion thereof (Fab′, Fab2′, etc.) or a receptor presented at thecell surface of a B or T cell lymphocyte, and which is able, by saidbinding, to induce an immune response. The term “T cell epitope” in thecontext of the present invention refers to a dominant, sub-dominant orminor T cell epitope, i.e. a part of an antigenic protein that isspecifically recognised and bound by a receptor at the cell surface of aT lymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognised by Tcells and able to activate them, among all the possible T cell epitopesof a protein.

The T cell epitope is an epitope recognised by MHC class II molecules,which consists of a sequence of +/−9 amino acids which fit in the grooveof the MHC II molecule. Within a peptide sequence representing a T cellepitope, the amino acids in the epitope are numbered P1 to P9, aminoacids N-terminal of the epitope are numbered P− 1, P− 2 and so on, aminoacids C terminal of the epitope are numbered P+ 1, P+ 2 and so on.Peptides recognised by MHC class II molecules and not by MHC class Imolecules are referred to as MHC class II restricted T cell epitopes.

The term “MHC” refers to “major histocompatibility antigen”. In humans,the MHC genes are known as HLA (“human leukocyte antigen”) genes.Although there is no consistently followed convention, some literatureuses HLA to refer to HLA protein molecules, and MHC to refer to thegenes encoding the HLA proteins. As such the terms “MHC” and “HLA” areequivalents when used herein. The HLA system in man has its equivalentin the mouse, i.e., the H2 system. The most intensely-studied HLA genesare the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C,HLA-DPA1, HLA-DPB1, HLA-DQA1, HLAs DOB1, HLA-DRA, and HLA-DRB1. Inhumans, the MHC is divided into three regions: Class I, II, and Ill. TheA, B, and C genes belong to MHC class I, whereas the six D genes belongto class II. MHC class I molecules are made of a single polymorphicchain containing 3 domains (alpha 1, 2 and 3), which associates withbeta 2 microglobulin at cell surface. Class II molecules are made of 2polymorphic chains, each containing 2 chains (alpha 1 and 2, and beta 1and 2).

Class I MHC molecules are expressed on virtually all nucleated cells.

Peptide fragments presented in the context of class I MHC molecules arerecognised by CD8+ T lymphocytes (cytolytic T lymphocytes or CTLs). CD8+T lymphocytes frequently mature into cytolytic effectors which can lysecells bearing the stimulating antigen. Class II MHC molecules areexpressed primarily on activated lymphocytes and antigen-presentingcells. CD4+ T lymphocytes (helper T lymphocytes or Th) are activatedwith recognition of a unique peptide fragment presented by a class IIMHC molecule, usually found on an antigen-presenting cell like amacrophage or dendritic cell. CD4+ T lymphocytes proliferate and secretecytokines such as IL-2, IFN-gamma and IL-4 that supportantibody-mediated and cell mediated responses.

Functional HLAs are characterised by a deep binding groove to whichendogenous as well as foreign, potentially antigenic peptides bind. Thegroove is further characterised by a well-defined shape andphysico-chemical properties. HLA class I binding sites are closed, inthat the peptide termini are pinned down into the ends of the groove.They are also involved in a network of hydrogen bonds with conserved HLAresidues. In view of these restraints, the length of bound peptides islimited to 8-10 residues. However, it has been demonstrated thatpeptides of up to 12 amino acid residues are also capable of binding HLAclass I. Comparison of the structures of different HLA complexesconfirmed a general mode of binding wherein peptides adopt a relativelylinear, extended conformation, or can involve central residues to bulgeout of the groove.

In contrast to HLA class I binding sites, class II sites are open atboth ends. This allows peptides to extend from the actual region ofbinding, thereby “hanging out” at both ends. Class II HLAs can thereforebind peptide ligands of variable length, ranging from 9 to more than 25amino acid residues. Similar to HLA class I, the affinity of a class IIligand is determined by a “constant” and a “variable” component. Theconstant part again results from a network of hydrogen bonds formedbetween conserved residues in the HLA class II groove and the main-chainof a bound peptide. However, this hydrogen bond pattern is not confinedto the N- and C-terminal residues of the peptide but distributed overthe whole chain. The latter is important because it restricts theconformation of complexed peptides to a strictly linear mode of binding.This is common for all class II allotypes. The second componentdetermining the binding affinity of a peptide is variable due to certainpositions of polymorphism within class II binding sites. Differentallotypes form different complementary pockets within the groove,thereby accounting for subtype-dependent selection of peptides, orspecificity. Importantly, the constraints on the amino acid residuesheld within class II pockets are in general “softer” than for class I.There is much more cross reactivity of peptides among different HLAclass II allotypes. The sequence of the +/−9 amino acids of an MHC classII T cell epitope that fit in the groove of the MHC II molecule areusually numbered P1 to P9. Additional amino acids N-terminal of theepitope are numbered P− 1, P− 2 and so on, amino acids C-terminal of theepitope are numbered P+ 1, P+ 2 and so on.

The term “homologue” as used herein with reference to the epitopes usedin the context of the invention, refers to molecules having at least50%, at least 70%, at least 80%, at least 90%, at least 95% or at least98% amino acid sequence identity with the naturally occurring epitope,thereby maintaining the ability of the epitope to bind an antibody orcell surface receptor of a B and/or T cell. Particular homologues of anepitope correspond to the natural epitope modified in at most three,more particularly in at most 2, most particularly in one amino acid.

The term “derivative” as used herein with reference to the peptides ofthe invention refers to molecules which contain at least the peptideactive portion (i.e. capable of eliciting cytolytic CD4+ T cellactivity) and, in addition thereto comprises a complementary portionwhich can have different purposes such as stabilising the peptides oraltering the pharmacokinetic or pharmacodynamic properties of thepeptide.

The term “sequence identity” of two sequences as used herein relates tothe number of positions with identical nucleotides or amino acidsdivided by the number of nucleotides or amino acids in the shorter ofthe sequences, when the two sequences are aligned. In particular, thesequence identity is from 70% to 80%, from 81% to 85%, from 86% to 90%,from 91% to 95%, from 96% to 100%, or 100%.

The terms “peptide-encoding polynucleotide (or nucleic acid)” and“polynucleotide (or nucleic acid) encoding peptide” as used herein referto a nucleotide sequence, which, when expressed in an appropriateenvironment, results in the generation of the relevant peptide sequenceor a derivative or homologue thereof. Such polynucleotides or nucleicacids include the normal sequences encoding the peptide, as well asderivatives and fragments of these nucleic acids capable of expressing apeptide with the required activity. The nucleic acid encoding a peptideaccording to the invention or fragment thereof is a sequence encodingthe peptide or fragment thereof originating from a mammal orcorresponding to a mammalian, most particularly a human peptidefragment.

The term “immune disorders” or “immune diseases” refers to diseaseswherein a reaction of the immune system is responsible for or sustains amalfunction or non-physiological situation in an organism. Included inimmune disorders are, inter alia, allergic disorders and autoimmunediseases.

The terms “allergic diseases” or “allergic disorders” as used hereinrefer to diseases characterised by hypersensitivity reactions of theimmune system to specific substances called allergens (such as pollen,stings, drugs, or food). Allergy is the ensemble of signs and symptomsobserved whenever an atopic individual patient encounters an allergen towhich he has been sensitised, which may result in the development ofvarious diseases, in particular respiratory diseases and symptoms suchas bronchial asthma. Various types of classifications exist and mostlyallergic disorders have different names depending upon where in themammalian body it occurs. “Hypersensitivity” is an undesirable(damaging, discomfort-producing and sometimes fatal) reaction producedin an individual upon exposure to an antigen to which it has becomesensitised; “immediate hypersensitivity” depends of the production ofIgE antibodies and is therefore equivalent to allergy.

The terms “autoimmune disease” or “autoimmune disorder” refer todiseases that result from an aberrant immune response of an organismagainst its own cells and tissues due to a failure of the organism torecognise its own constituent parts (down to the sub-molecular level) as“self’. The group of diseases can be divided in two categories,organ-specific and systemic diseases. An “allergen” is defined as asubstance, usually a macromolecule or a proteic composition whichelicits the production of IgE antibodies in predisposed, particularlygenetically disposed, individuals (atopics) patients. Similardefinitions are presented in Liebers et al. (1996) Clin. Exp. Allergy26, 494-516.

The term “therapeutically effective amount” refers to an amount of thepeptide of the invention or derivative thereof, which produces thedesired therapeutic or preventive effect in a patient. For example, inreference to a disease or disorder, it is the amount which reduces tosome extent one or more symptoms of the disease or disorder, and moreparticularly returns to normal, either partially or completely, thephysiological or biochemical parameters associated with or causative ofthe disease or disorder. Typically, the therapeutically effective amountis the amount of the peptide of the invention or derivative thereof,which will lead to an improvement or restoration of the normalphysiological situation. For instance, when used to therapeuticallytreat a mammal affected by an immune disorder, it is a daily amountpeptide/kg body weight of the said mammal. Alternatively, where theadministration is through gene-therapy, the amount of naked DNA or viralvectors is adjusted to ensure the local production of the relevantdosage of the peptide of the invention, derivative or homologue thereof.

The term “natural” when referring to a peptide relates to the fact thatthe sequence is identical to a fragment of a naturally occurring protein(wild type or mutant). In contrast therewith the term “artificial”refers to a sequence which as such does not occur in nature. Anartificial sequence is obtained from a natural sequence by limitedmodifications such as changing/deleting/inserting one or more aminoacids within the naturally occurring sequence or by adding/removingamino acids N- or C-terminally of a naturally occurring sequence.

In this context, it is realised that peptide fragments are generatedfrom antigens, typically in the context of epitope scanning. Bycoincidence such peptides may comprise in their sequence an MHC class IIepitope and in their proximity a sequence with the modified redox motifH-X(0,2)-C-X(2)-[CST] [SEQ ID NO: 78, 90 or 91] or [CST]-X(2)-C-X(0,2)-H[SEQ ID NO: 79, 92 or 93]. Herein “proximity” means that between MHCclass II epitope sequence and between the above H-X(0,2)-C-X(2)-[CST][SEQ ID NO: 78, 90 or 91] or [CST]-X(2)-C-X(0,2)-H [SEQ ID NO: 79, 92 or93] motifs, there can be an amino acid sequence of at most 7 aminoacids, at most 4 amino acids, at most 2 amino acids, or even 0 aminoacids (in other word epitope and motif sequence are immediately adjacentto each other).

Accordingly, specific embodiments of the present invention excludepeptide fragments of antigens which accidentally comprise as well an MHCclass T cell and a redox motif sequence immediately adjacent to eachother or separated by an amino acid sequence of up to 2, 4 or 7 aminoacids.

Other specific embodiments of the present invention exclude peptidefragments of antigens which accidentally comprise as well an MHC classII T cell epitope and a redox motif sequence, regardless from thespacing between epitope and motif modified redox motif.

Peptide fragments of antigens are studied for the immunogenic propertiesbut are generally not used a therapeutic agent (apart from the field ofallergy and tumour vaccination). Thus in the absence of any knowledge ofthe improved properties of the peptides of the present invention the useof such peptides as medicaments is unprecedented

Amino acids are referred to herein with their full name, theirthree-letter abbreviation or their one letter abbreviation.

Motifs of amino acid sequences are written herein according to theformat of Prosite. Motifs are used to describe a certain sequencevariety at specific parts of a sequence. The symbol X is used for aposition where any amino acid is accepted.

Alternatives are indicated by listing the acceptable amino acids for agiven position, between square brackets (‘[ ]’). For example: [CST]stands for an amino acid selected from Cys, Ser or Thr. Amino acidswhich are excluded as alternatives are indicated by listing them betweencurly brackets (‘{ }’). For example: {AM} stands for any amino acidexcept Ala and Met. The different elements in a motif are separated fromeach other by a hyphen -. Repetition of an identical element within amotif can be indicated by placing behind that element a numerical valueor a numerical range between parentheses. For example: X(2) correspondsto X-X; X(2, 5) corresponds to 2, 3, 4 or 5 X amino acids, A(3)corresponds to A-A-A.

Thus, H-C-X(2)-C [SED ID NO:80] can be written as HCXXC [SED ID NO:80].Equally C-X(2)-C-X(0,2) represents the three possibilities wherein thereis between H and C, none, one or two amino acids; namely CXXCH [SEQ IDNO:83], CXXCXH [SEQ ID NO:94] and CXXCXXH [SEQ ID NO:95].

Equally H-X(0,2)-C-X(2)-C represents the three possibilities whereinthere is between H and C, none, one or two amino acids. namely HCXXC[SEQ ID NO:80], HXCXXC [SEQ ID NO:96] and HXXCXXC [SEQ ID NO:97].

To distinguish between the amino acids X, those between H and C arecalled external amino acids X (single underlined in the above sequence),those within the redox motif are called internal amino acids X (doubleunderlined in the above sequence).

X represents any amino acid, particularly an L-amino acid, moreparticularly one of the 20 naturally occurring L-amino acids.

A peptide, comprising a T cell epitope and a modified peptide motifsequence, having reducing activity is capable of generating a populationof antigen-specific cytolytic CD4+ T cell towards antigen-presentingcells.

Accordingly, in its broadest sense, the invention relates to peptideswhich comprise at least one T-cell epitope of an antigen (self ornon-self) with a potential to trigger an immune reaction, and a modifiedthioreductase sequence motif with a reducing activity on peptidedisulfide bonds. The T cell epitope and the modified redox motifsequence may be immediately adjacent to each other in the peptide oroptionally separated by a one or more amino acids (so called linkersequence). Optionally the peptide additionally comprises an endosometargeting sequence and/or additional “flanking” sequences.

The peptides of the invention comprise an MHC class II T-cell epitope ofan antigen (self or non-self) with a potential to trigger an immunereaction, and a modified redox motif. The reducing activity of the motifsequence in the peptide can be assayed for its ability to reduce asulfhydryl group such as in the insulin solubility assay wherein thesolubility of insulin is altered upon reduction, or with afluorescence-labelled substrate such as insulin. An example of suchassay is described in more detail in the experimental section of thisapplication.

The modified redox motif may be positioned at the amino-terminus side ofthe T-cell epitope or at the carboxy-terminus of the T-cell epitope.

Peptide fragments with reducing activity are encountered inthioreductases which are small disulfide reducing enzymes includingglutaredoxins, nucleoredoxins, thioredoxins and other thiol/disulfideoxydoreductases (Holmgren (2000) Antioxid. Redox Signal. 2, 811-820;Jacquot et al. (2002) Biochem. Pharm. 64, 1065-1069). They aremultifunctional, ubiquitous and found in many prokaryotes andeukaryotes. They exert reducing activity for disulfide bonds on proteins(such as enzymes) through redox active cysteines within conserved activedomain consensus sequences: C-X(2)-C [SEQ ID NO:71], C-X(2)-S [SEQ IDNO:72], C-X(2)-T [SEQ ID NO:73], S-X(2)-C [SEQ ID NO:74], T-X(2)-C [SEQID NO:75] (Fomenko et al. (2003) Biochemistry 42, 11214-11225; Fomenkoet al. (2002) Prot. Science 11, 2285-2296), in which X stands for anyamino acid. Such domains are also found in larger proteins such asprotein disulfide isomerase (PDI) and phosphoinositide-specificphospholipase C.

The 4 amino acid redox motif as known from e.g. Fomenko andWO2008/017517 comprises a cysteine at position 1 and/or 4; thus themotif is either C-X(2)-[CST][SEQ ID NO:77] or [CST]-X(2)-C [SEQ IDNO:76]. Such a tetrapeptide sequence will be referred to as “the motif’.The motif in a peptide can be any of the alternatives C-X(2)-C [SEQ IDNO:71], S-X(2)-C [SEQ ID NO:74], T-X(2)-C [SEQ ID NO:75], C-X(2)-S [SEQID NO:72] or C-X(2)-T [SEQ ID NO:73]. In particular, peptides containthe sequence motif C-X(2)-C [SEQ ID NO:71].

The “modified” redox motif of the peptides of the present inventiondiffers from the prior art in that immediately adjacent cysteine andoutside the motif a Histidine is present, in other words the modifiedredox motif is written as H-X(0,2)-C-X(2)-[CST] [SEQ ID NO: 78, 90 or91] or [CST]-X(2)-C-X(0,2)-H [SEQ ID NO: 79, 92 or 93].

Embodiments hereof are H-XX-C-X(2)-[CST] [SEQ ID NO: 91],H-X-C-X(2)-[CST][SEQ ID NO: 90], H-C-X(2)-[CST] [SEQ ID NO:78],[CST]-X(2)-C-XX-H [SEQ ID NO: 93] [CST]-X(2)-C-X-H [SEQ ID NO:92], and[CST]-X(2)-C-H [SEQ ID NO:79],

More specific embodiments are

H-C-X(2)-S [SEQ ID NO:81], H-X-C-X(2)-S [SEQ ID NO:98], H-XX-C-X(2)-S[SEQ ID NO: 99], H-C-X(2)-T [SEQ ID NO:82], H-X-C-X(2)-T [SEQ ID NO:100], H-XX-C-X(2)-T [SEQ ID NO: 101], S-X(2)-C-H [SEQ ID NO:84],S-X(2)-C-X-H [SEQ ID NO:102] S-X(2)-CXX-H [SEQ ID NO:103] T-X(2)-C-H[SEQ ID NO:85], T-X(2)-C-X-H [SEQ ID NO: 104], T-X(2)-C-XX-H [SEQ ID NO:105], C-X(2)-C-H [SEQ ID NO:83], C-X(2)-C-X-H [SEQ ID NO:94],C-X(2)-C-XX-H [SEQ ID NO:95], H-C-X(2)-C [SEQ ID NO:80], H-X-C-X(2)-C[SEQ ID NO:96], H-XX-C-X(2)-C [SEQ ID NO:97].

In specific embodiments of the invention peptides with a H-C-X(2)-C-H[SEQ ID NO:86] motif are excluded from the scope of the invention.

Other specific embodiments are peptides wherein a cysteine amino acid ofthe redox motif is flanked by two histidine sequences such as HCHxC [SEQID NO:106] or CxxHCH [SEQ ID NO:107]

As explained in detail further on, the peptides of the present inventioncan be made by chemical synthesis, which allows the incorporation ofnon-natural amino acids. Accordingly, “C” in the above recited redoxmodified redox motifs represents either cysteine or another amino acidswith a thiol group such as mercaptovaline, homocysteine or other naturalor non-natural amino acids with a thiol function. In order to havereducing activity, the cysteines present in a modified redox motifshould not occur as part of a cystine disulfide bridge. Nevertheless, aredox modified redox motif may comprise modified cysteines such asmethylated cysteine, which is converted into cysteine with free thiolgroups in vivo. X can be any of the 20 natural amino acids, including S,C, or T or can be a non-natural amino acid. In particular embodiments Xis an amino acid with a small side chain such as Gly, Ala, Ser or Thr.In further particular embodiments, X is not an amino acid with a bulkyside chain such as Trp. In further particular embodiments X is notCysteine. In further particular embodiments at least one X in themodified redox motif is His. In other further particular embodiments atleast one X in the modified redox is Pro.

Peptides may further comprise modifications to increase stability orsolubility, such as modification of the N-terminal NH₂ group or the Cterminal COOH group (e.g. modification of the COOH into a CONH₂ group).

In the peptides of the present invention comprising a modified redoxmotif, the motif is located such that, when the epitope fits into theMHC groove, the motif remains outside of the MHC binding groove. Themodified redox motif is placed either immediately adjacent to theepitope sequence within the peptide [in other words a linker sequence ofzero amino acids between motif and epitope], or is separated from the Tcell epitope by a linker comprising an amino acid sequence of 7 aminoacids or less. More particularly, the linker comprises 1, 2, 3, or 4amino acids. Specific embodiments are peptides with a 0, 1 or 2 aminoacid linker between epitope sequence and modified redox motif sequence.Alternatively, a linker may comprise 5, 6, 7, 8, 9 or 10 amino acids. Inthose peptides where the modified redox motif sequence is adjacent tothe epitope sequence this is indicated as position P− 4 to P− 1 or P+ 1to P+ 4 compared to the epitope sequence. Apart from a peptide linker,other organic compounds can be used as linker to link the parts of thepeptide to each other (e.g. the modified redox motif sequence to the Tcell epitope sequence).

The peptides of the present invention can further comprise additionalshort amino acid sequences N or C-terminally of the sequence comprisingthe T cell epitope and the modified redox motif. Such an amino acidsequence is generally referred to herein as a ‘flanking sequence’. Aflanking sequence can be positioned between the epitope and an endosomaltargeting sequence and/or between the modified redox motif and anendosomal targeting sequence. In certain peptides, not comprising anendosomal targeting sequence, a short amino acid sequence may be presentN and/or C terminally of the modified redox motif and/or epitopesequence in the peptide. More particularly a flanking sequence is asequence of between 1 and 7 amino acids, most particularly a sequence of2 amino acids.

The modified redox motif may be located N-terminal from the epitope.

In certain embodiments, wherein the modified redox motif contains onecysteine, this cysteine is present in the modified redox motif in theposition remote from the epitope, thus the modified redox motif occursfor example as H-C-X(2)-T [SEQ ID NO:82] or H-C-X(2)-S [SEQ ID NO:81]N-terminally of the epitope or occurs as T-X(2)-C-H [SEQ ID NO:85] orS-X(2)-C-H [SEQ ID NO:84] C-terminally of the epitope.

In certain embodiments of the present invention, peptides are providedcomprising one epitope sequence and a modified redox motif sequence. Infurther particular embodiments, the modified redox motif occurs severaltimes (1, 2, 3, 4 or even more times) in the peptide, for example asrepeats of the modified redox motif which can be spaced from each otherby one or more amino acids or as repeats which are immediately adjacentto each other. Alternatively, one or more modified redox motifs areprovided at both the N and the C terminus of the T cell epitopesequence.

Other variations envisaged for the peptides of the present inventioninclude peptides which contain repeats of a T cell epitope sequencewherein each epitope sequence is preceded and/or followed by themodified redox motif (e.g. repeats of “modified redox motif-epitope” orrepeats of “modified redox motif-epitope-modified redox motif’). Hereinthe modified redox motifs can all have the same sequence but this is notobligatory. It is noted that repetitive sequences of peptides whichcomprise an epitope which in itself comprises the modified redox motifwill also result in a sequence comprising both the ‘epitope’ and a‘modified redox motif’.

In such peptides, the modified redox motif within one epitope sequencefunctions as a modified redox motif outside a second epitope sequence.

Typically the peptides of the present invention comprise only one T cellepitope. As described below a T cell epitope in a protein sequence canbe identified by functional assays and/or one or more in silicaprediction assays. The amino acids in a T cell epitope sequence arenumbered according to their position in the binding groove of the MHCproteins. A T-cell epitope present within a peptide consist of between 8and 25 amino acids, yet more particularly of between 8 and 16 aminoacids, yet most particularly consists of 8, 9, 10, 11, 12, 13, 14, 15 or16 amino acids.

In a more particular embodiment, the T cell epitope consists of asequence of 9 amino acids. In a further particular embodiment, theT-cell epitope is an epitope, which is presented to T cells by MHC-classII molecules [MHC class II restricted T cell epitopes]. Typically T cellepitope sequence refers to the octapeptide or more specificallynonapeptide sequence which fits into the cleft of an MHC II protein.

The T cell epitope of the peptides of the present invention cancorrespond either to a natural epitope sequence of a protein or can be amodified version thereof, provided the modified T cell epitope retainsits ability to bind within the MHC cleft, similar to the natural T cellepitope sequence. The modified T cell epitope can have the same bindingaffinity for the MHC protein as the natural epitope, but can also have alowered affinity. In particular, the binding affinity of the modifiedpeptide is no less than 10-fold less than the original peptide, moreparticularly no less than 5 times less. Peptides of the presentinvention have a stabilising effect on protein complexes. Accordingly,the stabilising effect of the peptide-MHC complex compensates for thelowered affinity of the modified epitope for the MHC molecule.

The sequence comprising the T cell epitope and the reducing compoundwithin the peptide can be further linked to an amino acid sequence (oranother organic compound) that facilitates uptake of the peptide intolate endosomes for processing and presentation within MHC class IIdeterminants. The late endosome targeting is mediated by signals presentin the cytoplasmic tail of proteins and correspond to well-identifiedpeptide motifs such as the dileucine-based [DE]XXXL[LI] [SEQ ID NO:87]or DXXLL [SEQ ID NO:88] motif, the tyrosine-based YXXΦ [SEQ ID NO:89]motif or the so called acidic cluster motif. The symbol Φ representsamino acid residues with a bulky hydrophobic side chains such as Phe,Tyr and Trp. The late endosome targeting sequences allow for processingand efficient presentation of the antigen-derived T cell epitope byMHC-class II molecules. Such endosomal targeting sequences arecontained, for example, within the gp75 protein (Vijayasaradhi et al.(1995) J. Cell. Biol. 130, 807-820), the human CD3 gamma protein, theHLA-BM 11 (Copier et al. (1996) J. Immunol. 157, 1017-1027), thecytoplasmic tail of the DEC205 receptor (Mahnke et al. (2000) J. CellBiol. 151, 673-683). Other examples of peptides which function assorting signals to the endosome are disclosed in the review of Bonifacioand Traub (2003) Annu. Rev. Biochem. 72, 395-447. Alternatively, thesequence can be that of a subdominant or minor T cell epitope from aprotein, which facilitates uptake in late endosome without overcomingthe T cell response towards the antigen. The late endosome targetingsequence can be located either at the amino-terminal or at thecarboxy-terminal end of the antigen derived peptide for efficient uptakeand processing and can also be coupled through a flanking sequence, suchas a peptide sequence of up to 10 amino acids. When using a minor T cellepitope for targeting purpose, the latter is typically located at theamino-terminal end of the antigen derived peptide.

Accordingly, the present invention envisages peptides of antigenicproteins and their use in eliciting specific immune reactions. Thesepeptides can either correspond to fragments of proteins which comprise,within their sequence i.e. a reducing compound and a T cell epitopeseparated by at most 10, preferably 7 amino acids or less.Alternatively, and for most antigenic proteins, the peptides of theinvention are generated by coupling a reducing compound, moreparticularly a reducing modified redox motif as described herein,N-terminally or C-terminally to a T cell epitope of the antigenicprotein (either directly adjacent thereto or with a linker of at most10, more particularly at most 7 amino acids). Moreover the T cellepitope sequence of the protein and/or the modified redox motif can bemodified and/or one or more flanking sequences and/or a targetingsequence can be introduced (or modified), compared to the naturallyoccurring sequence. Thus, depending on whether or not the features ofthe present invention can be found within the sequence of the antigenicprotein of interest, the peptides of the present invention can comprisea sequence which is ‘artificial’ or ‘naturally occurring’.

The peptides of the present invention can vary substantially in length.The length of the peptides can vary from 13 or 14 amino acids, i.e.consisting of an epitope of 8-9 amino acids, adjacent thereto themodified redox motif 5 amino acids with the histidine, up to 20, 25, 30,40, 50, 75, 100 or 200 amino acids. For example, a peptide may comprisean endosomal targeting sequence of 40 amino acids, a flanking sequenceof about 2 amino acids, a motif as described herein of 5 amino acids, alinker of 4 amino acids and a T cell epitope peptide of 9 amino acids.

Accordingly, in particular embodiments, the complete peptides consist ofbetween 13 amino acids up to 50, 75, 100 or 200 amino acids. Moreparticularly, where the reducing compound is a modified redox motif asdescribed herein, the length of the (artificial or natural) sequencecomprising the epitope and modified redox motif optionally connected bya linker (referred to herein as ‘epitope-modified redox motif’sequence), without the endosomal targeting sequence, is critical. The‘epitope-modified redox motif’ more particularly has a length of 13, 14,15, 16, 17, 18 or 19 amino acids. Such peptides of 13 or 14 to 19 aminoacids can optionally be coupled to an endosomal targeting signal ofwhich the size is less critical.

As detailed above, in particular embodiments, the peptides of thepresent invention comprise a reducing modified redox motif as describedherein linked to a T cell epitope sequence.

A small number of protein sequences, fragments of proteins or syntheticpeptides may by coincidence comprise a modified redox motif sequence.However the chance that these proteins comprise a MHC class T cellepitope in the proximity of the modified redox sequence becomes verysmall. If existing such peptides will be probably known from epitopescanning experiments wherein sets of overlapping peptide fragments aresynthesised. In such publications the interest goes to the epitope andneglect the relevance of a modified redox motif with a Histidine and therelevance of such peptides in medical applications.

Such peptides are thus accidental disclosures unrelated to the inventiveconcept of the present invention.

In further particular embodiments, the peptides of the invention arepeptides comprising T cell epitopes which do not comprise an amino acidsequence with redox properties within their natural sequence.

However, in alternative embodiments, the T cell epitope may comprise anysequence of amino acids ensuring the binding of the epitope to the MHCcleft.

Where an epitope of interest of an antigenic protein comprises amodified redox motif such as described herein within its epitopesequence, the immunogenic peptides according to the present inventioncomprise the sequence of a modified redox motif as described hereinand/or of another reducing sequence coupled N- or C-terminally to theepitope sequence such that (contrary to the modified redox motif presentwithin the epitope, which is buried within the cleft) the attachedmodified redox motif can ensure the reducing activity.

Accordingly the T cell epitope and motif are immediately adjacent orseparated from each other and do not overlap. To assess the concept of“immediately adjacent” or “separated”, the 8 or 9 amino acid sequencewhich fits in the MHC cleft is determined and the distance between thisoctapeptide or nonapeptide with the modified redox motif pentapeptide isdetermined.

Generally, the peptides of the present invention are not natural (thusno fragments of proteins as such) but artificial peptides which contain,in addition to a T cell epitope, a modified redox motif as describedherein, whereby the modified redox motif is immediately separated fromthe T cell epitope by a linker consisting of up to seven, mostparticularly up to four or up to 2 amino acids.

It has been shown that upon administration (i.e. injection) to a mammalof a peptide according to the invention (or a composition comprisingsuch a peptide), the peptide elicits the activation of T cellsrecognising the antigen derived T cell epitope and provides anadditional signal to the T cell through reduction of surface receptor.This supra-optimal activation results in T cells acquiring cytolyticproperties for the cell presenting the T cell epitope, as well assuppressive properties on bystander T cells. In this way, the peptidesor composition comprising the peptides described in the presentinvention, which contain an antigen-derived T cell epitope and, outsidethe epitope, a modified redox motif can be used for direct immunisationof mammals, including human beings. The invention thus provides peptidesof the invention or derivatives thereof, for use as a medicine.Accordingly, the present invention provides therapeutic methods whichcomprise administering one or more peptides according to the presentinvention to a patient in need thereof.

The present invention offers methods by which antigen-specific T cellsendowed with cytolytic properties can be elicited by immunisation withsmall peptides. It has been found that peptides which contain (i) asequence encoding a T cell epitope from an antigen and (ii) a consensussequence with redox properties, and further optionally also comprising asequence to facilitate the uptake of the peptide into late endosomes forefficient MHC-class II presentation, elicit suppressor T-cells.

The immunogenic properties of the peptides of the present invention areof particular interest in the treatment and prevention of immunereactions. Peptides described herein are used as medicament, moreparticularly used for the manufacture of a medicament for the preventionor treatment of an immune disorder in a mammal, more in particular in ahuman.

The present invention describes methods of treatment or prevention of animmune disorder of a mammal in need for such treatment or prevention, byusing the peptides of the invention, homologues or derivatives thereof,the methods comprising the step of administering to said mammalsuffering or at risk of an immune disorder a therapeutically effectiveamount of the peptides of the invention, homologues or derivativesthereof such as to reduce the symptoms of the immune disorder. Thetreatment of both humans and animals, such as, pets and farm animals isenvisaged. In an embodiment the mammal to be treated is a human. Theimmune disorders referred to above are in a particular embodimentselected from allergic diseases and autoimmune diseases. Allergicdiseases are conventionally described as type-1 mediated diseases orIgE-mediated diseases. Clinical manifestations of allergic diseasesinclude bronchial asthma, allergic rhinitis, atopic dermatitis, foodhypersensitivity and anaphylactic reactions to insect bites or drugs.Allergic diseases are caused by hypersensitivity reactions of the immunesystem to specific substances called allergens (such as pollen, stings,drugs, or food). The most severe form of an allergic disorder isanaphylactic shock, which is a medical emergency. Allergens includeairborne allergens, such as those of house dust mite, pets and pollens.Allergens also include ingested allergens responsible for foodhypersensitivity, including fruits, vegetables and milk. In order totreat the above diseases, peptides according to the invention aregenerated from the antigenic proteins or allergens known or believed tobe a causative factor of the disease. The allergens that can be used forselection of T-cell epitopes are typically allergens which are selectedfrom the group consisting of: food allergens present in peanuts, fishe.g. codfish, egg white, crustacean e.g. shrimp, milk e.g. cow's milk,wheat, cereals, fruits of the Rosacea family (apple, plum, strawberry),vegetables of the Liliacea, Cruciferae, Solanaceae and Umbelliferaefamilies, tree nuts, sesame, peanut, soybean and other legume familyallergens, spices, melon, avocado, mango, fig, banana, . . . house dustmites allergens obtained from Dermatophagoides spp or D. pteronyssinus,D. farinae and D. microceras, Euroglyphus maynei or Blomia sp.,allergens from insects present in cockroach or Hymenoptera, allergensfrom pollen, especially pollens of tree, grass and weed, allergens fromanimals, especially in cat, dog, horse and rodent, allergens from fungi,especially from Aspergillus, Altemaria or Cladosporium, and occupationalallergens present in products such as latex, amylase, etc.

As an example on allergens, in the context of the present invention thederp 2 peptide CGFSSNYCQIYPPNANKIR [SEQ ID NO:9] is modified inHCGFSSNYCQIYPPNANKIR [SEQ ID NO:10] or HCGFCSNYCQIYPPNANKIR [SEQ IDNO:11]. As a further example on allergens the der p 2 peptideCHGSEPCIIHRGKPF [SEQ ID NO:12], is modified into HCHGSEPCIIHRGKPF [SEQID NO:13], HCHGCEPCIIHRGKPF [SEQ ID NO:14] more typically intoHCxGSEPCIIHRGKPF [SEQ ID NO:15] or HCxGCEPCIIHRGKPF wherein x is not Hisor Cys [SEQ ID NO:16].

As a further example on allergens the Beta lactoglobulin peptideCHGCAQKKIIAEK [SEQ ID NO:17] is modified into HCHGCAQKKIIAEK [SEQ IDNO:18], more typically into HCxGCAQKKIIAEK, wherein x is not Cys or His[SEQ ID NO:19].

As an example on auto-immune disease the thyroid peroxidase peptideCGPCMNEELTERL [SEQ ID NO:20] is modified into HCGPCMNEELTERL [SEQ IDNO:21].

As an example on auto-immune disease the thyroglobulin peptideCGPSAALTWVQTH [SEQ ID NO:22] is modified into HCGPCAALTWVQTH [SEQ IDNO:23].

The present invention further relates to peptides with the modifiedredox motif comprising MHC class II T cell epitopes of viral proteinswhich are encoded by the backbone of viral vectors used in gene therapyand gene vaccination. The present invention further relates to methodsof treatment or prevention of immunogenic response against a viralvector. Examples of viral vectors (e.g. from adenovirus,adeno-associated virus, herpes virus or poxvirus, retroviruses orlentivirus) and viral proteins (e.g. capsid protein) are disclosed inWO2009101204.

As an example of the teaching of the present invention, the adenoviralpeptide CHGCPTLLYVLFEV [SEQ ID NO:24] is modified into HCHGCPTLLYVLFEV[SEQ ID NO:25], more typical HCxGCPTLLYVLFEV wherein X is not Cys or His[SEQ ID NO:26]

As a further example, adenoviral late protein 2 peptide CGPCGGYVPFHIQVP[SEQ ID NO:27] is modified into HCGPCGGYVPFHIQVP [SEQ ID NO:28].

The present invention further relates to peptides with the modifiedredox motif comprising MHC class II T cell epitopes of proteins ofintracellular pathogens. The present invention further relates tomethods of treatment and prevention of infections with intracellularpathogens. Examples of intracellular pathogens (viruses [DNA vs RNAviruses, ss vs ds viruses, bacteria, mycobacteria or parasites with anintracellular life cycle) and antigens are discussed in WO2009101208(for example Herpesviridae, Flaviviridae and Picornaviridae, influenza,measles and immunodeficiency viruses, papilloviruses. Bacteria andmycobacteria including Mycobacterium tuberculosis, and othermycobacteria pathogenic for humans or animals such as Yersiniae,Brucellae, Chlamydiae, Mycoplasmae, Rickettsiae, Salmonellae andShigellae. Parasites include Plasmodiums, Leishmanias, Trypanosom as,Toxoplasma gondii, Listeria sp., Histoplasma sp.

As a further example the CSP antigen of malaria CGHCDKHIEQYLK [SEQ IDNO:29]. is modified into HCGHCDKHIEQYLK [SEQ ID NO:30], more typicalinto HCGxCDKHIEQYLK, wherein x is not Cys or His [SEQ ID NO:31].

As a further example the CGHCEKKICKMEK [SEQ ID NO:32]. peptide of thesame antigen is modified into HCGHCEKKICKMEK [SEQ ID NO:33], moretypically into HCGxCEKKICKMEK [SEQ ID NO:34], wherein x is not Cys orHis.

As a further example the peptide from influenza hemagglutinin ismodified from CGHCKYVKQNTLK [SEQ ID NO:35] into HCGHCKYVKQNTLK [SEQ IDNO:36], more typically into HCGxCKYVKQNTLK, wherein x is not Cys or His[SEQ ID NO:37].

As a further example the peptide from Leishmania Lack antigenCGHCEHPIVVSGS [SEQ ID NO:38] is modified into HCGHCEHPIVVSGS [SEQ IDNO:39], more typical HCGxCEHPIVVSGS, wherein X is not Cys or His [SEQ IDNO:40].

As a further example the peptide of the gp120 subunit of the Env proteinof HIV, is modified from CGHCRAMYAPPIA [SEQ ID NO:41] intoHCGHCRAMYAPPIA [SEQ ID NO:42], more typically into HCGxCRAMYAPPIA,wherein x is not Cys or His [SEQ ID NO:43].

The present invention further relates to peptides with the modifiedredox motif comprising MHC class II T cell epitopes of solubleallofactors such as used in replacement therapies. The present inventionfurther relates to methods of treatment and prevention of immunereactions against soluble allofactors.

Examples of soluble allofactors are disclosed in WO2009101206.

As an example of the present invention the peptide ofcomplementarity-determining region (CDR) 3 of the VH region of theB02C11 antibody, against factor VIII, CHGCYCAVPDDPDA [SEQ ID NO:44], ismodified into HCHGCYCAVPDDPDA [SEQ ID NO:45], more typically intoHCxGCYCAVPDDPDA, wherein x is not Cys or His [SEQ ID NO:46].

As a further example the peptide derived from another anti-Factor VIIIantibody, CGHCGGIRLHPTHYSIR [SEQ ID NO:47] is modified intoHCGHCGGIRLHPTHYSIR [SEQ ID NO:48], more typically intoHCGxCGGIRLHPTHYSIR wherein x is not Cys or His [SEQ ID NO:49].

The present invention further relates to peptides with the modifiedredox motif comprising MHC class II T cell epitopes of tumour associatedantigens. The present invention further relates to methods of treatmentand prevention of tumours. Examples of relevant tumours (e.g. oncogene,proto-oncogene, viral protein, a surviving factor, clonotypicdeterminant) and tumour associated antigens are disclosed in WOWO2009101205. Such tumor associated antigens include viral antigens oftumour causing viruses such as HPV, tumour associated antigens of apatient which have a wild-type sequence but have an increased expressionin tumours, or antigens which have a mutated sequence by pointmutations, deletions, frame shifts, or chromosomal rearrangements.

As an example of the teaching of the present invention the MAGE-3peptide CHGCYRQVPGSDP [SEQ ID NO:50] is modified into HCHGCYRQVPGSDP[SEQ ID NO:51], more typical into HCxGCYRQVPGSDP wherein x is not Cys orHis [SEQ ID NO:52].

As a further example the cyclin D peptide CHGCFVALCATDV [SEQ ID NO:53]is modified into HCHGCFVALCATDV [SEQ ID NO:54], more typical intoHCxGCFVALCATDV, wherein X is not Cys or His [SEQ ID NO:55].

As a further example the surviving peptide CHGCFKELEGWEP [SEQ ID NO:56]is modified into HCHGCFKELEGWEP [SEQ ID NO:57], more typical intoHCxGCFKELEGWEP wherein X is not Cys or His [SEQ ID NO:58].

As a further example the Epstein Barr virus peptide CHGCVASSYAAAQ [SEQID NO:59] is modified into HCHGCVASSYAAAQ [SEQ ID NO:60], more typicalinto HCxGCVASSYAAAQ wherein X is not Cys or His [SEQ ID NO:61].

The present invention further relates to peptides with the modifiedredox motif comprising MHC class II T cell epitopes of alloantigenicprotein of an allograft. The present invention further relates tomethods of treatment and prevention of allograft rejection. Examples arebone marrow grafts, solid organ grafts such as kidney, lung, heart,liver, pancreas, bone or skin, or cellular grafts such as cord bloodcell graft, stem cell graft, or pancreatic islet cell grafts. Examplesof alloantigenic proteins are disclosed in WO2009100505, such as minorhistocompatibility antigens, major histocompatibility antigens ortissue-specific antigens.

As an example of the present invention, the peptide from murine Dbyantigen CHGCFNSNRANSS [SEQ ID NO:62] is modified into HCHGCFNSNRANSS[SEQ ID NO:63], more particular into HCxGCFNSNRANSS wherein x is not Cysor His [SEQ ID NO: 64].

In another example the sequence from human Dby CGHCLVLAPTREL [SEQ IDNO:65], is modified into HCGHCLVLAPTREL [SEQ ID NO:66], moreparticularly into HCGxCLVLAPTREL, wherein x is not Cys or His [SEQ IDNO:67].

In another example the murine Black 6 strain specific peptideCGHCPEFLEQKRA [SEQ ID NO:68] is modified into HCGHCPEFLEQKRA [SEQ IDNO:69], more typically into HCGxCPEFLEQKRA, wherein x is not Cys or His[SEQ ID NO:70].

For all the above peptides additional variant are envisaged, whereinbetween Histidine and Cysteine, one or two amino acids X are present.Typically these external amino acid(s) X is (are) not His, Cys, Ser orThr.

The peptides of the present invention can also be used in diagnostic invitro methods for detecting class II restricted CD4+ T cells in asample. In this method a sample is contacted with a complex of an MHCclass II molecule and a peptide according to the present invention. TheCD4+ T cells detected by measuring the binding of the complex with cellsin the sample, wherein the binding of the complex to a cell isindicative for the presence of CD4+ T cells in the sample.

The complex can be a fusion protein of the peptide and an MHC class IImolecule.

Alternatively MHC molecules in the complex are tetramers. The complexcan be provided as a soluble molecule or can be attached to a carrier.

The T cell epitope corresponding to an antigenic protein (or immunogen)suitable for use in the context of the present invention is typically auniversal or promiscuous T cell epitope (i.e. a T cell epitope capableof binding to a majority of the MHC class II molecules), moreparticularly present upon an airborne allergen or a foodborne allergen.In particular embodiments, the allergen is selected from the groupconsisting of rhino-sinusitis allergens, allergic bronchial asthmaallergens and atopic dermatitis allergens. Allergens can also be mainallergens present in moulds or various drugs such as hormones,antibiotics, enzymes, etc. (See also the definition in Clin. Exp.Allergy 26, 494-516 (1996) and in Molecular Biology of Allergy andImmunology, Ed. R. Bush (1996)). Other allergens related to specificallergic diseases are also well known in the art and can be found on theinternet, e.g. on www.allergome.org.

Autoimmune diseases are broadly classified into two categories,organ-specific and systemic diseases. The precise aetiology of systemicauto-immune diseases is not identified. In contrast, organ-specificauto-immune diseases are related to a specific immune response includingB and T cells, which targets the organ and thereby induces and maintainsa chronic state of local inflammation. Examples of organ-specificauto-immune diseases include type 1 diabetes, myasthenia gravis,thyroiditis and multiple sclerosis. In each of these conditions, asingle or a small number of auto-antigens have been identified,including insulin, the acetylcholine muscle receptor, thyroid peroxidaseand major basic protein, respectively. It is well recognised thatsuppression of this organ-specific immune response is beneficial andleads to partial or complete recovery of organ function. There is,however, no therapy, which would suppress such an immune response in anantigen-specific manner. Current therapy rather makes use ofnon-specific suppression obtained by the use of corticosteroids andimmunosuppressive agents, all exhibiting significant side-effectsrelated to their absence of specificity, thereby limiting their use andtheir overall efficacy. A non-limiting list of examples of organspecific autoimmune disorders and auto-antigens involved therein whichare envisaged within the context of the present invention are:

-   thyroid diseases: thyroglobulin, thyroid peroxidase, TSH receptor-   type 1 diabetes: insulin (proinsulin), glutamic acid decarboxylase    (GAD), tyrosine phosphatase IA-2, heat-shock protein HSP65,    islet-specific glucose6-phosphatase catalytic subunit related    protein (IGRP)-   Adrenalitis: 21-OH hydroxylase-   polyendocrine syndromes: 17-alpha hydroxylase, histidine    decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase-   gastritis & pernicious anemia: H+/K+ ATPase intrinsic factor-   multiple sclerosis: myelin oligodendrocyte glycoprotein (MOG),    myelin basic protein (MBP), proteolipid (PLP)-   myasthenia gravis: acetyl-choline receptor-   ocular diseases: retinol-binding protein (RBP)-   inner ear diseases: type II and type IX collagen-   celiac disease: tissue transglutaminase-   inflammatory bowel diseases: pANCA histone H1 protein-   Atherosclerosis: heat-shock protein HSP60

According to the present invention, immunogenic peptides are providedwhich comprise a T-cell epitope of an antigen (self or non-self) with apotential to trigger an immune reaction. In a particular embodiment, theT-cell epitope is a dominant T-cell epitope.

Accordingly, in particular embodiments, the methods of treatment andprevention of the present invention comprise the administration of animmunogenic peptide as described herein, wherein the peptide comprise aT-cell epitope of an antigenic protein which plays a role in the diseaseto be treated (for instance such as those described above). In furtherparticular embodiments, the epitope used is a dominant epitope.

The present invention further relates to methods to produce peptideswith a MHC class II T cell epitope and a modified redox motif.

In a first step the method comprises the step of providing the sequenceof an antigenic protein of interest and identifying an MHC class II Tcell epitope sequence in the antigen. Epitope sequences may have beendescribed yet for the antigenic protein under consideration.Alternatively they are determined by in silico methods, in vitro methodsor in vivo methods. In addition the antigenic protein is screened forthe presence of the modified redox motif, which requires no specific insilico methods.

There is a very small, but existing, chance that an antigenic proteincontains within its sequence a H-X(0,2)C-X(2)-[CST] [SEQ ID NO:78, 90 or91] or [CST]-X(2)-C-X(0,2)-H [SEQ ID NO:79, 92 or 93] motif in the closeproximity of a T cell epitope sequence (i.e. separated from the T cellepitope by 7 or less amino acids). If so, a fragment of the antigenicprotein comprising T cell epitope and motif can be used for the methodsand uses of the prevent invention. The epitope in such proteins may havebeen discussed in the prior art but the presence, let alone, therelevance of such modified redox motif is not discussed. There has beenaccordingly no incentive in the prior art to select such peptidefragments, or to use such peptide fragments for the methods describedherein. In certain embodiments, wherein the peptide is based on afragment of a protein which contains an MHC class II T cell epitope anda modified redox motif such a peptide sequence may be further modifiedby changing the length of the sequence between the epitope and themodified redox motif, changing amino acids in the linker sequence,changing a Ser or Thr in the motif into a Cysteine or changing aminoacids at one or both X positions within the motif.

Other antigenic proteins which are used for the design of peptides maycontain a H-X(0,2)-C-X(2)-[CST] [SEQ ID NO:78, 90 or 91] or[CST]-X(2)-C-X(0,2)-H [SEQ ID NO:79, 92 or 93] sequence in its sequencewhich is further remote from a MHC class II T cell epitope (more than 7amino acids from the epitope sequence).

In such cases a peptide can be produced wherein only the distancebetween the epitope and the motif is shortened and whereby the sequenceof the motif and neighbouring amino acids are preserved. If deemedsuitable, amino acids outside the motif, Serine or threonine in themotif or one or both X positions are changed. More general, antigenicproteins which are used for the design of peptides will not contain aH-X(0,2)-C-X(2)-[CST] [SEQ ID NO:78, 90 or 91] or [CST]-X(2)-C-X(0,2)-H[SEQ ID NO:79, 92 or 93] sequence within their protein sequence.

Peptides in accordance of the present invention will be prepared bysynthesising a peptide wherein T cell epitope and modified redox motifwill be separated by 0 to 7 amino acids. In certain embodiments themodified redox motif can be obtained by introducing 1, 2 or 3 mutationsoutside the epitope sequence, to preserve the sequence context asoccurring in the protein. Typically amino-acids in P− 2 and P− 1, aswell as in P+ 10 and P+ 11, with reference to the nonapeptide which arepart of the natural sequence are preserved in the peptide sequence.These flanking residues generally stabilize the binding to MHC class II.In other embodiments the sequence N terminal or C terminal of theepitope will be unrelated to the sequence of the antigenic proteincontaining the T cell epitope sequence.

In other specific embodiments, peptides are prepared by modifyingpeptides with a T cell epitope and a C-X(2)-[CST] [SEQ ID NO:77] or[CST]-X(2)-C [SEQ ID NO:76] motif as disclosed in WO2008/017517.Addition of a Histidine or modification of an amino acid into aHistidine leads to peptides of the present invention with aH-X(2,0)-C-X(2)-[CST] [SEQ ID NO:78, 90 or 91] or [CST]-X(2)-C-X(0,2)-H[SEQ ID NO:79, 92 or 93] sequence.

Thus based upon the above methods for designing a peptide, a peptide isgenerated by chemical peptide synthesis, recombinant expression methodsor in more exceptional cases, proteolytic or chemical fragmentation ofproteins.

Peptides as produced in the above methods can be tested for the presenceof a T cell epitope in in vitro and in vivo methods, can be tested fortheir reducing activity in in vitro assays. As a final quality control,the peptides can be tested in in vitro assays to verify whether thepeptides can generated CD4+ T cells which are cytolytic via an apoptoticpathway for antigen presenting cells presenting the antigen whichcontains the epitope sequence which is also present in the peptide withthe modified redox motif.

The identification and selection of a T-cell epitope from antigenicproteins, for use in the context of the present invention is known to aperson skilled in the art.

To identify an epitope suitable for use in the context of the presentinvention, isolated peptide sequences of an antigenic protein are testedby, for example, T cell biology techniques, to determine whether thepeptide sequences elicit a T cell response. Those peptide sequencesfound to elicit a T cell response are defined as having T cellstimulating activity.

Human T cell stimulating activity can further be tested by culturing Tcells obtained from an individual sensitive to e.g. a mite allergen,(i.e. an individual who has an IgE mediated immune response to a miteallergen) with a peptide/epitope derived from the allergen anddetermining whether proliferation of T cells occurs in response to thepeptide/epitope as measured, e.g., by cellular uptake of tritiatedthymidine. Stimulation indices for responses by T cells topeptides/epitopes can be calculated as the maximum CPM in response to apeptide/epitope divided by the control CPM. A T cell stimulation index(S.I.) equal to or greater than two times the background level isconsidered “positive.” Positive results are used to calculate the meanstimulation index for each peptide/epitope for the group ofpeptides/epitopes tested.

Non-natural (or modified) T-cell epitopes can further optionally betested on their binding affinity to MHC class II molecules. This can beperformed in different ways.

For instance, soluble HLA class II molecules are obtained by lysis ofcells homozygous for a given class II molecule. The latter is purifiedby affinity chromatography. Soluble class II molecules are incubatedwith a biotin-labelled reference peptide produced according to itsstrong binding affinity for that class II molecule. Peptides to beassessed for class II binding are then incubated at differentconcentrations and their capacity to displace the reference peptide fromits class II binding is calculated by addition of neutravidin. Methodscan be found in for instance Texier et al., (2000) J. Immunology 164,3177-3184.) According to the present invention, the immunogenicproperties of T cell epitopes are increased by linking it to themodified redox motif which has enhance reducing properties.Particularly, peptides of the present invention comprising at least oneT cell epitope and the modified redox motif as described herein have amean T cell stimulation index of greater than or equal to 2.0. A peptidehaving a T cell stimulation index of greater than or equal to 2.0 isconsidered useful as a therapeutic agent. More particularly, peptidesaccording to the invention have a mean T cell stimulation index of atleast 2.5, at least 3.5, at least 4.0, or even at least 5.0. Inaddition, peptides have typically a positivity index (P.I.) of at leastabout 100, at least 150, at least about 200 or at least about 250. Thepositivity index for a peptide is determined by multiplying the mean Tcell stimulation index by the percent of individuals, in a population ofindividuals with an immune response (eg sensitive to house dust mite)(e. g., at least 9 individuals, at least 16 individuals or at least 29or 30, or even more), who have T cells that respond to the peptide (thuscorresponding to the SI multiplied by the promiscuous nature of thepeptide/epitope). Thus, the positivity index represents both thestrength of a T cell response to a peptide (S.I.) and the frequency of aT cell response to a peptide in a population of individuals with animmune response (e.g.) sensitive to house dust mite.

In order to determine optimal T cell epitopes by, for example, finemapping techniques, a peptide having T cell stimulating activity andthus comprising at least one T cell epitope as determined by T cellbiology techniques is modified by addition or deletion of amino acidresidues at either the amino- or carboxyterminus of the peptide andtested to determine a change in T cell reactivity to the modifiedpeptide. If two or more peptides which share an area of overlap in thenative protein sequence are found to have human T cell stimulatingactivity, as determined by T cell biology techniques, additionalpeptides can be produced comprising all or a portion of such peptidesand these additional peptides can be tested by a similar procedure.Following this technique, peptides are selected and producedrecombinantly or synthetically. T cell epitopes or peptides are selectedbased on various factors, including the strength of the T cell responseto the peptide/epitope (e.g., stimulation index) and the frequency ofthe T cell response to the peptide in a population of individuals.

Additionally and/or alternatively, one or more in vitro algorithms canbe used to identify a T cell epitope sequence within an antigenicprotein. Suitable algorithms include, but are not limited to thosedescribed in Zhang et al. (2005) Nucleic Acids Res 33, W180-W183(PREDBALB); Salomon & Flower (2006) BMC Bioinformatics 7, 501 (MHCBN);Schuler et al. (2007) Methods Mol. Biol. 409, 75-93 (SYFPEITHI); Donnes& Kohlbacher (2006) Nucleic Acids Res. 34, W194-W197 (SVMHC); Kolaskar &Tongaonkar (1990) FEBS Lett. 276, 172-174, Guan et al. (2003) Appl.Bioinformatics 2, 63-66 (MHCPred) and Singh and Raghava (2001)Bioinformatics 17, 1236-1237 (Propred).

More particularly, such algorithms allow the prediction within anantigenic protein of one or more octa- or nonapeptide sequences whichwill fit into the groove of an MHC II molecule and this for differentHLA types.

The peptides of the present invention can be generated using recombinantDNA techniques, in bacteria, yeast, insect cells, plant cells ormammalian cells. In view of the limited length of the peptides, they canbe prepared by chemical peptide synthesis, wherein peptides are preparedby coupling the different amino acids to each other. Chemical synthesisis particularly suitable for the inclusion of e.g. D-amino acids, aminoacids with non-naturally occurring side chains or natural amino acidswith modified side chains such as methylated cysteine.

Chemical peptide synthesis methods are well described and peptides canbe ordered from companies such as Applied Biosystems and othercompanies.

Peptide synthesis can be performed as either solid phase peptidesynthesis (SPPS) or contrary to solution phase peptide synthesis. Thebest-known SPPS methods are t-Boc and Fmoc solid phase chemistry:

During peptide synthesis several protecting groups are used. For examplehydroxyl and carboxyl functionalities are protected by t-butyl group,lysine and tryptophan are protected by t-Boc group, and asparagine,glutamine, cysteine and histidine are protected by trityl group, andarginine is protected by the pbf group. If appropriate, such protectinggroups can be left on the peptide after synthesis. Peptides can belinked to each other to form longer peptides using a ligation strategy(chemoselective coupling of two unprotected peptide fragments) asoriginally described by Kent (Schnelzer & Kent (1992) Int. J. Pept.Protein Res. 40, 180-193) and reviewed for example in Tam et al. (2001)Biopolymers 60, 194-205 provides the tremendous potential to achieveprotein synthesis which is beyond the scope of SPPS. Many proteins withthe size of 100-300 residues have been synthesised successfully by thismethod. Synthetic peptides have continued to play an ever increasingcrucial role in the research fields of biochemistry, pharmacology,neurobiology, enzymology and molecular biology because of the enormousadvances in the SPPS.

Alternatively, the peptides can be synthesised by using nucleic acidmolecules which encode the peptides of this invention in an appropriateexpression vector which include the encoding nucleotide sequences. SuchDNA molecules may be readily prepared using an automated DNA synthesiserand the well-known codon-amino acid relationship of the genetic code.Such a DNA molecule also may be obtained as genomic DNA or as cDNA usingoligonucleotide probes and conventional hybridisation methodologies.Such DNA molecules may be incorporated into expression vectors,including plasmids, which are adapted for the expression of the DNA andproduction of the polypeptide in a suitable host such as bacterium, e.g.Escherichia coli, yeast cell, animal cell or plant cell.

The physical and chemical properties of a peptide of interest (e.g.solubility, stability) are examined to determine whether the peptideis/would be suitable for use in therapeutic compositions. Typically thisis optimised by adjusting the sequence of the peptide. Optionally, thepeptide can be modified after synthesis (chemical modifications e.g.adding/deleting functional groups) using techniques known in the art.

T cell epitopes on their own are thought to trigger early events at thelevel of the T helper cell by binding to an appropriate HLA molecule onthe surface of an antigen presenting cell and stimulating the relevant Tcell subpopulation. These events lead to T cell proliferation,lymphokine secretion, local inflammatory reactions, the recruitment ofadditional immune cells to the site, and activation of the B cellcascade leading to production of antibodies. One isotype of theseantibodies, IgE, is fundamentally important in the development ofallergic symptoms and its production is influenced early in the cascadeof events, at the level of the T helper cell, by the nature of thelymphokines secreted. A T cell epitope is the basic element or smallestunit of recognition by a T cell receptor where the epitope comprisesamino acid residues essential to receptor recognition, which arecontiguous in the amino acid sequence of the protein.

However, upon administration of the peptides with a T-cell epitope and aredox motif, the following events are believed to happen:

activation of antigen (i) specific T cells resulting from cognateinteraction with the antigen-derived peptide presented by MHC-class IImolecules;the reductase sequence reduces T cell surface proteins, such as the CD4molecule. the second domain of which contains a constrained disulfidebridge. This transduces a signal into T cells. Among a series ofconsequences related to increased oxidative pathway, important eventsare increased calcium influx and translocation of the NF-kBtranscription factor to the nucleus. The latter results in increasedtranscription of IFN-gamma and granzymes, which allows cells to acquirecytolytic properties via an apoptosis-inducing mechanism; the cytolyticproperty affects cells presenting the peptide by a mechanism, whichinvolves granzyme B secretion, and Fas-FasL interactions. Since the cellkilling effect is obtained via an apoptotic pathway, cytolic cells is amore appropriate term for these cells than cytotoxic cells. Destructionof the antigen-presenting target cells prevents activation of other Tcells specific for epitopes located on the same antigen, or to anunrelated antigen that would be processed by the same antigen-presentingcell; an additional consequence of T cell activation is to suppressactivation of bystander T cells by a cell-cell contact dependentmechanism. In such a case, T cells activated by an antigen presented bya different antigen-presenting cell is also suppressed provided bothcytolytic and bystander T cells are in close proximity, namely activatedon the surface of the same antigen-presenting cell.

The above-postulated mechanism of action is substantiated withexperimental data disclosed in the above cited PCT application andpublications of the present inventor.

The present invention provides methods for generating antigen-specificcytolytic CD4+ T cells either in vivo or in vitro and, independentlythereof, methods to discriminate cytolytic CD4+ T cells from other cellpopulations such as Foxp3+ Tregs based on characteristic expressiondata.

The present invention describes in vivo methods for the production ofthe antigen-specific CD4+ T cells. A particular embodiment relates tothe method for producing or isolating the CD4+ T cells by immunisinganimals (including humans) with the peptides of the invention asdescribed herein and then isolating the CD4+ T cells from the immunisedanimals. The present invention describes in vitro methods for theproduction of antigen specific cytolytic CD4+ T cells towards APC. Thepresent invention provides methods for generating antigen specificcytolytic CD4+ T cells towards APC.

In one embodiment, methods are provided which comprise the isolation ofperipheral blood cells, the stimulation of the cell population in vitroby an immunogenic peptide according to the invention and the expansionof the stimulated cell population, more particularly in the presence ofIL-2. The methods according to the invention have the advantage a highnumber of CD4+ T cells is produced and that the CD4+ T cells can begenerated which are specific for the antigenic protein (by using apeptide comprising an antigen-specific epitope).

In an alternative embodiment, the CD4+ T cells can be generated in vivo,i.e. by the injection of the immunogenic peptides described herein to asubject, and collection of the cytolytic CD4+ T cells generated in vivo.

The antigen-specific cytolytic CD4+ T cells towards APC, obtainable bythe methods of the present invention are of particular interest for theadministration to mammals for immunotherapy, in the prevention ofallergic reactions and the treatment of auto-immune diseases. Both theuse of allogenic and autogeneic cells are envisaged.

Cytolytic CD4+ T cells populations are obtained as described hereinbelow. Antigen-specific cytolytic CD4+ T cells as described herein canbe used as a medicament, more particularly for use in adoptive celltherapy, more particularly in the treatment of acute allergic reactionsand relapses of autoimmune diseases such as multiple sclerosis. Isolatedcytolytic CD4+ T cells or cell populations, more particularlyantigen-specific cytolytic CD4+ T cell populations generated asdescribed are used for the manufacture of a medicament for theprevention or treatment of immune disorders. Methods of treatment byusing the isolated or generated cytolytic CD4+ T cells are disclosed.

As explained in WO2008/017517 cytolytic CD4+ T cells towards APC can bedistinguished from natural Treg cells based on expressioncharacteristics of the cells. More particularly, a cytolytic CD4+ T cellpopulation demonstrates one or more of the following characteristicscompared to a natural Treg cell population: an increased expression ofsurface markers including CD103, CTLA-4, FasI and ICOS upon activation,

intermediate expression of CD25,expression of CD4, ICOS, CTLA-4, GITR and low or no expression of CD127(IL7-R), no expression of CD27.expression of transcription factor T-bet and egr-2 (Krox-20) but not ofthe transcription repressor Foxp3,a high production of IFN-gamma and no or only trace amounts of IL-10,IL-4, IL-5, IL-13 or TGF-beta.

Further the cytolytic T cells express CD45RO and/or CD45RA, do notexpress CCR7, CD27 and present high levels of granzyme B and othergranzymes as well as Fas ligand.

The peptides of the invention will, upon administration to a livinganimal, typically a human being, elicit specific T cells exerting asuppressive activity on bystander T cells.

This mechanism also implies and the experimental results show that thepeptides of the invention, although comprising a specific T-cell epitopeof a certain antigen, can be used for the prevention or treatment ofdisorders elicited by an immune reaction against other T-cell epitopesof the same antigen or in certain circumstances even for the treatmentof disorders elicited by an immune reaction against other T-cellepitopes of other different antigens if they would be presented throughthe same mechanism by MHC class II molecules in the vicinity of T cellsactivated by peptides of the invention.

Isolated cell populations of the cell type having the characteristicsdescribed above, which, in addition are antigen-specific, i.e. capableof suppressing an antigen-specific immune response are disclosed.

The peptides of the invention may also be used in gene therapy methodswell known in the art and the terminology used herein explaining the useof peptides according to the invention also includes the use of nucleicacids encoding or expressing immunogenic peptides according to theinvention.

The present invention describes nucleic acid sequences encoding thepeptides of the present invention and methods for their use. Differentmethods of achieving, by way of gene therapy, levels of peptides,homologues or derivatives thereof according to the invention in a mammalin vivo are envisaged within the context of the present invention.

Recombinant nucleic acid molecules encoding protein sequences can beused as naked DNA or in liposomes or other lipid systems for delivery totarget cells. Other methods for the direct transfer of plasmid DNA intocells are well known to those skilled in the art for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. In its simplest form, genetransfer can be performed by simply injecting minute amounts of DNA intothe nucleus of a cell, through a process of microinjection. Oncerecombinant genes are introduced into a cell, they can be recognised bythe cells normal mechanisms for transcription and translation, and agene product will be expressed. Other methods have also been attemptedfor introducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with calcium phosphate andtaken into cells by pinocytosis; electroporation, wherein cells areexposed to large voltage pulses to introduce holes into the membrane);lipofection/liposome fusion, wherein DNA is packed into lipophilicvesicles which fuse with a target cell; and particle bombardment usingDNA bound to small projectiles. Another method for introducing DNA intocells is to couple the DNA to chemically modified proteins.

Adenovirus proteins are capable of destabilising endosomes and enhancingthe uptake of DNA into cells. Mixing adenovirus to solutions containingDNA complexes, or the binding of DNA to polylysine covalently attachedto adenovirus using protein crosslinking agents substantially improvesthe uptake and expression of the recombinant gene. Adeno-associatedvirus vectors may also be used for gene delivery into vascular cells. Asused herein, “gene transfer” means the process of introducing a foreignnucleic acid molecule into a cell, which is commonly performed to enablethe expression of a particular product encoded by the gene.

The product may include a protein, polypeptide, anti-sense DNA or RNA,or enzymatically active RNA. Gene transfer can be performed in culturedcells or by direct administration into mammals. In another embodiment, avector comprising a nucleic acid molecule sequence encoding a peptideaccording to the invention is provided. In particular embodiments, thevector is generated such that the nucleic acid molecule sequence isexpressed only in a specific tissue. Methods of achievingtissue-specific gene expression are well known in the art. This can befor example achieved by placing the sequence encoding a peptideaccording to the invention under control of a promoter which directsexpression in one or more particular tissues.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, RNA virusesor bovine papilloma virus, may be used for delivery of nucleotidesequences (e.g., cDNA) encoding peptides, homologues or derivativesthereof according to the invention into the targeted tissues or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant viral vectors containing such codingsequences.

Accordingly, the present invention discloses the use of a nucleic acidwhich is capable of expressing the peptides of the invention, in vivo,for the treatment and/or prevention of diseases driven by an immuneresponse to a foreign or self antigen. According to one embodiment, thenucleic acid capable of expressing a peptide according to the inventionin vivo is a sequence encoding such a peptide, which is operably linkedto a promoter. Such a sequence can be administered directly orindirectly. For instance, an expression vector containing the codingsequence for a peptide according to the invention may be inserted intocells, after which the cells are grown in vitro and then injected orinfused into the patient. Alternatively the nucleic acid capable ofexpressing a peptide according to the invention in vivo is a sequencewhich modifies endogenous expression of the cells. The gene therapymethod may involve the use of an adenovirus vector including anucleotide sequence coding for peptides, homologues or derivativesthereof according to the invention or a naked nucleic acid moleculecoding for a peptide according to the invention. Alternatively,engineered cells containing a nucleic acid molecule coding for a peptideaccording to the invention may be injected.

Where the administration of one or more peptides according to theinvention is ensured through gene transfer (i.e. the administration of anucleic acid which ensures expression of peptides according to theinvention in vivo upon administration), the appropriate dosage of thenucleic acid can be determined based on the amount of peptide expressedas a result of the nucleic acid, such as e.g. by determining theconcentration of peptide in the blood after administration. Thus, in aparticular embodiment, the peptides of the invention are administeredthrough the use of polynucleotides encoding the peptides, whether in anexpression vector or not and thus the present invention also relates togene therapy methods. Another particular embodiment relates to the useof methods to induce a local overexpression of the peptides of theinvention for the treatment or prevention of immune disorders.

The present invention provides pharmaceutical compositions comprisingone or more peptides according to the present invention, furthercomprising a pharmaceutically acceptable carrier. As detailed above, thepresent invention also relates to the compositions for use as a medicineor to methods of treating a mammal of an immune disorder by using thecomposition and to the use of the compositions for the manufacture of amedicament for the prevention or treatment of immune disorders. Thepharmaceutical composition could for example be a vaccine suitable fortreating or preventing immune disorders, especially airborne andfoodborne allergy, as well as diseases of allergic origin. As an exampledescribed further herein of a pharmaceutical composition, a peptideaccording to the invention is adsorbed on an adjuvant suitable foradministration to mammals, such as aluminium hydroxide (alum).Typically, 50 μg of the peptide adsorbed on alum are injected by thesubcutaneous route on 3 occasions at an interval of 2 weeks. It shouldbe obvious for those skilled in the art that other routes ofadministration are possible, including oral, intranasal orintramuscular. Also, the number of injections and the amount injectedcan vary depending on the conditions to be treated. Further, otheradjuvants than alum can be used, provided they facilitate peptidepresentation in MHC-class II presentation and T cell activation.

Thus, while it is possible for the active ingredients to be administeredalone, they typically are presented as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the presentinvention comprise at least one active ingredient, as above described,together with one or more pharmaceutically acceptable carriers. Thepresent invention relates to pharmaceutical compositions, comprising, asan active ingredient, one or more peptides according to the invention,in admixture with a pharmaceutically acceptable carrier. Thepharmaceutical composition of the present invention should comprise atherapeutically effective amount of the active ingredient, such asindicated hereinafter in respect to the method of treatment orprevention. Optionally, the composition further comprises othertherapeutic ingredients. Suitable other therapeutic ingredients, as wellas their usual dosage depending on the class to which they belong, arewell known to those skilled in the art and can be selected from otherknown drugs used to treat immune disorders.

The term “pharmaceutically acceptable carrier” as used herein means anymaterial or substance with which the active ingredient is formulated inorder to facilitate its application or dissemination to the locus to betreated, for instance by dissolving, dispersing or diffusing thecomposition, and/or to facilitate its storage, transport or handlingwithout impairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the immunogenicpeptide in the composition. The pharmaceutically acceptable carrier maybe a solid or a liquid or a gas which has been compressed to form aliquid, i.e. the compositions of this invention can suitably be used asconcentrates, emulsions, solutions, granulates, dusts, sprays, aerosols,suspensions, ointments, creams, tablets, pellets or powders. Suitablepharmaceutical carriers for use in the pharmaceutical compositions andtheir formulation are well known to those skilled in the art, and thereis no particular restriction to their selection within the presentinvention. They may also include additives such as wetting agents,dispersing agents, stickers, adhesives, emulsifying agents, solvents,coatings, antibacterial and antifungal agents (for example phenol,sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodiumchloride) and the like, provided the same are consistent withpharmaceutical practice, i.e. carriers and additives which do not createpermanent damage to mammals. The pharmaceutical compositions of thepresent invention may be prepared in any known manner, for instance byhomogeneously mixing, coating and/or grinding the active ingredients, ina one-step or multi-steps procedure, with the selected carrier materialand, where appropriate, the other additives such as surface-activeagents. They may also be prepared by micronisation, for instance in viewto obtain them in the form of microspheres usually having a diameter ofabout 1 to 10 μm, namely for the manufacture of microcapsules forcontrolled or sustained release of the active ingredients.

Suitable surface-active agents, also known as emulgent or emulsifier, tobe used in the pharmaceutical compositions of the present invention arenon-ionic, cationic and/or anionic materials having good emulsifying,dispersing and/or wetting properties. Suitable anionic surfactantsinclude both water-soluble soaps and water-soluble syntheticsurface-active agents. Suitable soaps are alkaline or alkaline-earthmetal salts, unsubstituted or substituted ammonium salts of higher fattyacids (C10-C22), e.g. the sodium or potassium salts of oleic or stearicacid, or of natural fatty acid mixtures obtainable form coconut oil ortallow oil. Synthetic surfactants include sodium or calcium salts ofpolyacrylic acids; fatty sulphonates and sulphates; sulphonatedbenzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates orsulphates are usually in the form of alkaline or alkaline-earth metalsalts, unsubstituted ammonium salts or ammonium salts substituted withan alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. thesodium or calcium salt of lignosulphonic acid or dodecylsulphonic acidor a mixture of fatty alcohol sulphates obtained from natural fattyacids, alkaline or alkaline-earth metal salts of sulphuric or sulphonicacid esters (such as sodium lauryl sulphate) and sulphonic acids offatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazolederivatives typically contain 8 to 22 carbon atoms. Examples ofalkylarylsulphonates are the sodium, calcium or alcanolamine salts ofdodecyl benzene sulphonic acid or dibutyl-naphtalenesulphonic acid or anaphtalene-sulphonic acid/formaldehyde condensation product. Alsosuitable are the corresponding phosphates, e.g. salts of phosphoric acidester and an adduct of p-nonylphenol with ethylene and/or propyleneoxide, or phospholipids. Suitable phospholipids for this purpose are thenatural (originating from animal or plant cells) or syntheticphospholipids of the cephalin or lecithin type such as e.g.phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylglycerine,lysolecithin, cardio-lipin, dioctanylphosphatidylcholine,dipalmitoylphoshatidylcholine and their mixtures.

Suitable non-ionic surfactants include polyethoxylated andpoly-propoxylated derivatives of alkyl phenols, fatty alcohols, fattyacids, aliphatic amines or amides containing at least 12 carbon atoms inthe molecule, alkylarene sulphonates and dialkylsulphosuccinates, suchas polyglycol ether derivatives of aliphatic and cycloaliphaticalcohols, saturated and unsaturated fatty acids and alkylphenols, thederivatives typically containing 3 to 10 glycol ether groups and 8 to 20carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbonatoms in the alkyl moiety of the alkylphenol. Further suitable non-ionicsurfactants are water-soluble adducts of polyethylene oxide withpoylypropylene glycol, ethylenediamino-polypropylene glycol containing 1to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ethergroups. Such compounds usually contain from I to 5 ethyleneglycol unitsper propyleneglycol unit. Representative examples of non-ionicsurfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolicethers, polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethyleneglycol andoctylphenoxypolyethoxyethanol. Fatty acid esters of polyethylenesorbitan (such as polyoxyethylene sorbitan trioleate), glycerol,sorbitan, sucrose and pentaerythritol are also suitable non-ionicsurfactants. Suitable cationic surfactants include quaternary ammoniumsalts, particularly halides, having 4 hydrocarbon radicals optionallysubstituted with halo, phenyl, substituted phenyl or hydroxy; forinstance quaternary ammonium salts containing as N-substituent at leastone C8C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyland the like) and, as further substituents, unsubstituted or halogenatedlower alkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for thispurpose may be found for instance in “McCutcheon's Detergents andEmulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981),“Tensid-Taschenbucw’, 2 d ed. (Hanser Verlag, Vienna, 1981) and“Encyclopaedia of Surfactants, (Chemical Publishing Co., New York,1981). Peptides, homologues or derivatives thereof according to theinvention (and their physiologically acceptable salts or pharmaceuticalcompositions all included in the term “active ingredients”) may beadministered by any route appropriate to the condition to be treated andappropriate for the compounds, here the proteins and fragments to beadministered. Possible routes include regional, systemic, oral (solidform or inhalation), rectal, nasal, topical (including ocular, buccaland sublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intra-arterial, intrathecal andepidural). The preferred route of administration may vary with forexample the condition of the recipient or with the diseases to betreated. As described herein, the carrier(s) optimally are “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Theformulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraarterial,intrathecal and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. Such methods includethe step of bringing into association the active ingredient with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients.

Compressed tablets may be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein.

For local treatments for example on the skin, such as of the joint, theformulations are optionally applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),particularly 0.2 to 15% w/w and more particularly 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base. If desired, the aqueous phase of the cream base may include,for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas.

Examples of such dermal penetration enhancers include dimethylsulfoxideand related analogues. The oily phase of the emulsions of this inventionmay be constituted from known ingredients in a known manner. While thephase may comprise merely an emulsifier (otherwise known as anemulgent), it desirably comprises a mixture of at least one emulsifierwith a fat or an oil or with both a fat and an oil. Optionally, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabiliser, typically by including both an oil and afat.

Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus the cream should optionally be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, and particularly butylstearate, 2-ethylhexyl palmitate or a blend of branched chain estersknown as Crodamol CAP may be used. These may be used alone or incombination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used. Formulationssuitable for topical administration to the eye also include eye dropswherein the active ingredient is dissolved or suspended in a suitablecarrier, especially an aqueous solvent for the active ingredient. Theactive ingredient is optionally present in such formulations in aconcentration of 0.5 to 20%, advantageously 0.5 to 10% particularlyabout 1.5% w/w. Formulations suitable for topical administration in themouth include lozenges comprising the active ingredient in a flavouredbasis, usually sucrose and acacia or tragacanth; pastilles comprisingthe active ingredient in an inert basis such as gelatin and glycerine,or sucrose and acacia; and mouthwashes comprising the active ingredientin a suitable liquid carrier. Formulations for rectal administration maybe presented as a suppository with a suitable base comprising forexample cocoa butter or a salicylate. Formulations suitable for nasaladministration wherein the carrier is a solid include a coarse powderhaving a particle size for example in the range 20 to 500 microns(including particle sizes in a range between 20 and 500 microns inincrements of 5 microns such as 30 microns, 35 microns, etc), which isadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable formulations wherein the carrier is aliquid, for administration as for example a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol administration may be preparedaccording to conventional methods and may be delivered with othertherapeutic agents. Formulations suitable for vaginal administration maybe presented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Typical unit dosage formulations are those containing a daily dose orunit daily sub-dose, as herein above recited, or an appropriate fractionthereof, of an active ingredient. It should be understood that inaddition to the ingredients particularly mentioned above theformulations of this invention may include other agents conventional inthe art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents. Peptides, homologues or derivatives thereof according to theinvention can be used to provide controlled release pharmaceuticalformulations containing as active ingredient one or more compounds ofthe invention (“controlled release formulations”) in which the releaseof the active ingredient can be controlled and regulated to allow lessfrequency dosing or to improve the pharmacokinetic or toxicity profileof a given invention compound. Controlled release formulations adaptedfor oral administration in which discrete units comprising one or morecompounds of the invention can be prepared according to conventionalmethods. Additional ingredients may be included in order to control theduration of action of the active ingredient in the composition. Controlrelease compositions may thus be achieved by selecting appropriatepolymer carriers such as for example polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers,methylcellulose, carboxymethylcellulose, protamine sulfate and the like.The rate of drug release and duration of action may also be controlledby incorporating the active ingredient into particles, e.g.microcapsules, of a polymeric substance such as hydrogels, polylacticacid, hydroxymethylcellulose, polyniethyl methacrylate and the otherabove-described polymers. Such methods include colloid drug deliverysystems like liposomes, microspheres, microemulsions, nanoparticles,nanocapsules and so on. Depending on the route of administration, thepharmaceutical composition may require protective coatings.Pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation thereof. Typical carriers for this purpose therefore includebiocompatible aqueous buffers, ethanol, glycerol, propylene glycol,polyethylene glycol and the like and mixtures thereof. In view of thefact that, when several active ingredients are used in combination, theydo not necessarily bring out their joint therapeutic effect directly atthe same time in the mammal to be treated, the corresponding compositionmay also be in the form of a medical kit or package containing the twoingredients in separate but adjacent repositories or compartments. Inthe latter context, each active ingredient may therefore be formulatedin a way suitable for an administration route different from that of theother ingredient, e.g. one of them may be in the form of an oral orparenteral formulation whereas the other is in the form of an ampoulefor intravenous injection or an aerosol.

Cytolytic CD4+ T cells as obtained in the present invention, induce APCapoptosis after MHC-class II dependent cognate activation, affectingboth dendritic and B cells, as demonstrated in vitro and in vivo, and(2) suppress bystander T cells by a contact-dependent mechanism in theabsence of IL-10 and/or TGF-beta. Cytolytic CD4+ T cells can bedistinguished from both natural and adaptive Tregs, as discussed indetail in WO2008/017517.

The present invention will now be illustrated by means of the followingexamples which are provided without any limiting intention. Furthermore,all references described herein are explicitly included herein byreference.

EXAMPLES Example 1: Methodology to Assess Reducing Activity of Peptides

The reductase activity of the peptides is determined using a fluorescentdescribed in Tomazzolli et al. (2006) Anal. Biochem. 350, 105-112. Twopeptides with a FITC label become self-quenching when they covalentlyattached to each other via a disulfide bridge. Upon reduction by apeptide in accordance with the present invention, the reduced individualpeptides become fluorescent again.

Control experiments were performed with a peptide with a “normal”reducing peptide, i.e. a peptide with a redox motif but withoutadditional histidine and with a peptide comprising no redox motif.

Example 2: Determination of the Activation of Cells

Antigen specific cytolytic cells as obtained by the peptides of thepresent invention are capable to drive antigen presenting cells intoapoptosis. To evaluate the activation and prevent eventualover-activation of the cytolytic cells which would drive them themselvesin apoptosis, the phosphorylation status of Akt and Shp allows to draw acorrelation between activation of a cell (capable of apoptosis) andover-activation of a cell (self-apoptosis).

Example 3: Design of MOG Derived Peptides

An example of the peptides of the present invention is the peptide withsequence HCPYCSRVVHLYRNGKD [SEQ ID NO:1]. This peptide comprises theSRVVHLYRNGKD [SEQ ID NO:2] fragment of the human MOG protein (MyelinOligodendrocyte Glycoprotein)(uniprot 016653 accession number], whichitself contains the VVHLYRNGK nonapeptide MHC class II T cell epitopesequence [SEQ ID NO:3]. According to the definitions mentioned in theapplication, this 17 AA peptide comprises:

-   -   a modified redox motif H-C-X(2)-C [SEQ ID NO:80], with Pro and        Tyr as x,    -   a linker of 2 amino acids (Ser, Arg) between the motif and the T        cell epitope sequence,    -   a MHC class T cell epitope of nine amino acids with sequence        VVHLYRNGK [SEQ ID NO:3],    -   a one amino acid flanking sequence (Asp) c terminal of the        epitope.

Compared to the sequence of the MOG peptide fragment YRPPFSRVVHLYRNGKD[SEQ ID NO:4], YRPPF [SEQ ID NO:5] in the sequence has been replace bythe sequence HCPYC [SEQ ID NO:6].

Control peptides are:

YRPPFSRVVHLYRNGKD [SEQ ID NO:4], i.e. the above fragment of MOG.CPYCSRVVHLYRNGKD [SEQ ID NO:7], with a C(X)2C motif [SEQ ID NO:71] butlacking the additional histidine.SRVVHLYRNGKD [SEQ ID NO:2], lacking the also the C(X)2C motif [SEQ IDNO:71].

Peptides have been prepared by peptide synthesis with a CONH₂ modifiedcarboxyterminus and are tested for purity by mass spectrometry and HPLC.

Example 4: In Vitro Expansion of Cell Lines

Response of naive human CD4+ T cell lines towards peptides with a T cellepitope of MOG and a redox motif without (right bars FIG. 1) [SEQ IDNO:7] and with additional Histidine (left bars FIG. 1) [SEQ ID NO:1].

Equal amounts of both peptides were added to different naïve human CD4+T cell lines. The results represent cell number (as % of initial cellseeded) at the end of a clinical scale process leading to the productionof differentiated T cells with cytolytic properties.

This significant increase of cell conversion in vitro is thus obtainedwhen the added histidine is added, but only for cell lines of personspresenting the DR2 haplotype, and not for others haplotypes.

The use of such peptides is thus particularly interesting for the DR2+population (again 70% of the MS population) it seems that there is adefinite (and unexpected) advantage of using his-containing peptides.

Example 5: Use of a T Cell Epitope of MOG Protein in an In Vivo Modelfor Multiple Sclerosis

Multiple sclerosis can be induced in experimental models by immunisationwith the Myelin Oligodendrocyte Glycoprotein (MOG) peptide with a T cellepitope.

A group of C57BL/6 mice is adoptively transferred with a CD4+MOG-specific effector T cell clone following a protocol meant to inducea multiple sclerosis-like syndrome. This involves administration of theMOG peptide in complete Freund's adjuvant and 2 injections of Pertussistoxin. This protocol elicits an expansion of the effector T cell clone,which results in the development of signs compatible with multiplesclerosis within 12 days after the MOG peptide administration. A secondgroup of C57BL/6 mice is first adoptively transferred with aMOG-specific cytolytic T cell clone (obtained using the peptide with[SEQ ID NO:1]), followed after 1 day by the full protocol of diseaseinduction.

Peptides with SEQ ID NO:2, 4 and 7 are used as controls.

Example 6: Prevention and Suppression of Multiple Sclerosis

Groups of C57BL/6 mice are immunised subcutaneously (20 μg) with thepeptide of example 1 which contains the modified sequence motif [SEQ IDNO:1] or control peptide [SEQ ID NO:2, 4 or 7] adsorbed onto aluminiumhydroxide. Three injections are performed at 2-week intervals. Ten daysafter the last immunisation, mice are sacrificed and CD4+ T cells (2×10⁶cells) are prepared from the spleen using magnetic beads. CD4+ T cellsare then stimulated in vitro by the MOG T cell epitope (20 μg/ml)presented by adherent spleen cells (2×10⁶ cells).

After four re-stimulations, a T cell line is tested in a bystandersuppression assay with, as target cells, polyclonal CD4⁺CD25⁻ cellsobtained from animals in which EAE (Experimental autoimmuneencephalomyelitis) is effective. Only the cells obtained from animalsimmunised with the peptide with SEQ ID NO:1 and 7 containing the HC(X)2C[SEQ ID NO:80] or C(X)2C [SEQ ID NO:71] sequence motif have the capacityto induce death in target cells, as compared to the control consistingin effector CD4⁺CD25⁻ from EAE animals.

A group of C57BL/6 mice is adoptively transferred with a CD4+MOG-specific CD4+ T cell clone followed after 1 day by a protocol meantto induce a multiple sclerosis-like syndrome. This involvesadministration of the MOG peptide in complete Freund's adjuvant and 2injections of Pertussis toxin. This protocol elicits an expansion of theeffector T cell clone, which results in the development of signscompatible with multiple sclerosis within 12 days after the MOG peptideadministration. The clinical score developed by mice pre-treated with acytolytic T cell clone is compared to mice receiving only the fullprotocol of disease induction.

Example 7: Prevention of Multiple Sclerosis by Peptide Immunisation

In the model group, C57BL6 mice received, at day 0, SC injection of 100μg MOG peptide/400 μg Mycobacterium butyricum in CFA and ip injection of300 ng Bortetella pertussis in NaCl. At day +2, a second injection of B.pertussis is given. In the prevention group, C57BL/6 mice are immunisedby 5 injections with 20 μg of the peptide with SEQ ID NO:1, whichcontains the sequence motif HC(X)2C [SEQ ID NO:80], in IFA at 14 daysinterval before disease induction as in the model group. Controlexperiments are performed with the peptides with SEQ ID NO:2, 4 and 7.Scores are established as 0: no disease, 1: limp tail, 2: limp tail andloss of weight higher than 10%, 3: partial paralysis of hind limbs.

Example 9

Evaluation of Reductase Activity on a Synthetic Peptide

A FITC —NH-Gly-Cys-Asp-COOH peptide was synthesized (Eurogentec,Belgium) and self-quenched by solubilization in DMSO((FITC-Gly-Cys-Asp)_(ox)). The reduction of 2.5 μM(FITC-Gly-Cys-Asp)_(ox) was followed on a 96 well plate during 40minutes (25° C.) after incubation in PBS with peptide (25 μM) as listedin the accompanying table, or with 2 mM Dithiothreitol (DTT). Reductionwas measured as a function of increase in fluorescence read at 530 nmafter excitation at 494 nm, using a CytoFluor® multiplate reader(Applied Biosystems). Results are shown in the table of example 10 underthe heading “Reductase activity”.

Example 10

Polymerization of Human Recombinant CD4

Human recombinant CD4 (300 ng) is incubated in Hepes buffer with 50 μMof a peptide as listed in the Table for 15 minutes at 68° C. Fifty μM ofDTT is used as a positive control under the same conditions. LDS samplebuffer (7.5 μl; non-reducing) is then added to 15 μl of the peptide/CD4mixture. The mixture is then submitted to non-reducing PAGE. AfterCoomassie Blue staining, protein bands are analyzed for the presence ofmonomeric, dimeric or multimeric recCD4, as identified by the decreasedmigratory capacities into the gel. The Table indicates whether or not apolymerization had occurred (+).

Reductase CD4 Seq id activity polymer- no: N-term motif linker epitopeC-term (%) isation 108 H CPYC VRSLQP LALEGSLQK RG 68 + 109 HAA CPYCVRSLQP LALEGSLQK RG 0 + 110 AHA CPYC VRSLQP LALEGSLQK RG 13 + 111 AAACPYC VRSLQP LALEGSLQK RG 6 + 112 AAA CHPC VRSLQP LALEGSLQK RG 75 + 113AAH CHPC VRSLQP LALEGSLQK RG 64 + 114 AAA CHGC VRSLQP LALEGSLQK RG 22low

The Table provides various combinations of aminoacid sequences added atthe amino-terminal end of a class II-restricted epitope of humanproinsulin. These sequences are constituted of a amino-terminal sequence(N-term) in front of the first cysteine of the thioreductase-containingmotif, the motif itself, a linker, the epitope and the C-terminal end(C-term). Reductase activity is expressed in % as described inExample 1. The polymerization of human recombinant CD4 is measuredaccording to Example 2.

Peptides Disclosed in the Application.

In the below sequences 1-70, wherein x occurs, x is not cysteine or isnot histidine.

Overview of disclosed peptide sequences. HCPYCSRVVHLYRNGKD[SEQ ID NO: 1]      SRVVHLYRNGKD [SEQ ID NO: 2]        VVHLYRNGK[SEQ ID NO: 3] YRPPFSRVVHLYRNGKD [SEQ ID NO: 4] YRPPF [SEQ ID NO: 5]HCPYC [SEQ ID NO: 6]  CPYCSRVVHLYRNGKD [SEQ ID NO: 7] HCPYCSRVVHLYRNGK[SEQ ID NO: 8]  CGFSSNYCQIYPPNANKIR [SEQ ID NO: 9] HCGFSSNYCQIYPPNANKIR[SEQ ID NO: 10] HCGFCSNYCQIYPPNANKIR [SEQ ID NO: 11]  CHGSEPCIIHRGKPF[SEQ ID NO: 12] HCHGSEPCIIHRGKPF [SEQ ID NO: 13] HCHGCEPCIIHRGKPF[SEQ ID NO: 14] HCxGSEPCIIHRGKPF [SEQ ID NO: 15] HCxGCEPCIIHRGKPF[SEQ ID NO: 16]  CHGCAQKKIIAEK [SEQ ID NO: 17] HCHGCAQKKIIAEK[SEQ ID NO: 18] HCxGCAQKKIIAEK [SEQ ID NO: 19]  CGPCMNEELTERL[SEQ ID NO: 20] HCGPCMNEELTERL [SEQ ID NO: 21]  CGPSAALTWVQTH[SEQ ID NO: 22] HCGPSAALTWVQTH [SEQ ID NO: 23]  CHGCPTLLYVLFEV[SEQ ID NO: 24] HCHGCPTLLYVLFEV [SEQ ID NO: 25] HCxGCPTLLYVLFEV[SEQ ID NO: 26]  CGPCGGYVPFHIQVP [SEQ ID NO: 27] HCGPCGGYVPFHIQVP[SEQ ID NO: 28]  CGHCDKHIEQYLK [SEQ ID NO: 29] HCGHCDKHIEQYLK[SEQ ID NO: 30] HCGxCDKHIEQYLK [SEQ ID NO: 31]  CGHCEKKICKMEK[SEQ ID NO: 32] HCGHCEKKICKMEK [SEQ ID NO: 33] HCGxCEKKICKMEK[SEQ ID NO: 34]  CGHCKYVKQNTLK [SEQ ID NO: 35] HCGHCKYVKQNTLK[SEQ ID NO: 36] HCGxCKYVKQNTLK [SEQ ID NO: 37]  CGHCEHPIVVSGS[SEQ ID NO: 38] HCGHCEHPIVVSGS [SEQ ID NO: 39] HCGxCEHPIVVSGS[SEQ ID NO: 40]  CGHCRAMYAPPIA [SEQ ID NO: 41] HCGHCRAMYAPPIA[SEQ ID NO: 42] HCGxCRAMYAPPIA [SEQ ID NO: 43]  CHGCYCAVPDDPDA[SEQ ID NO: 44] HCHGCYCAVPDDPDA [SEQ ID NO: 45] HCxGCYCAVPDDPDA[SEQ ID NO: 46]  CGHCGGIRLHPTHYSIR [SEQ ID NO: 47] HCGHCGGIRLHPTHYSIR[SEQ ID NO: 48] HCGxCGGIRLHPTHYSIR [SEQ ID NO: 49]  CHGCYRQVPGSDP[SEQ ID NO: 50] HCHGCYRQVPGSDP [SEQ ID NO: 51] HCxGCYRQVPGSDP[SEQ ID NO: 52]  CHGCFVALCATDV [SEQ ID NO: 53] HCHGCFVALCATDV[SEQ ID NO: 54] HCxGCFVALCATDV [SEQ ID NO: 55]  CHGCFKELEGWEP[SEQ ID NO: 56] HCHGCFKELEGWEP [SEQ ID NO: 57] HCxGCFKELEGWEP[SEQ ID NO: 58]  CHGCVASSYAAAQ [SEQ ID NO: 59] HCHGCVASSYAAAQ[SEQ ID NO: 60] HCxGCVASSYAAAQ [SEQ ID NO: 61]  CHGCFNSNRANSS[SEQ ID NO: 62] HCHGCFNSNRANSS [SEQ ID NO: 63] HCxGCFNSNRANSS[SEQ ID NO: 64]  CGHCLVLAPTREL [SEQ ID NO: 65] HCGHCLVLAPTREL[SEQ ID NO: 66] HCGxCLVLAPTREL [SEQ ID NO: 67]  CGHCPEFLEQKRA[SEQ ID NO: 68] HCGHCPEFLEQKRA [SEQ ID NO: 69] HCGxCPEFLEQKRA[SEQ ID NO: 70] CXXC [SEQ ID NO: 71] CXXS [SEQ ID NO: 72] CXXT[SEQ ID NO: 73] SXXC [SEQ ID NO: 74] TXXC [SEQ ID NO: 75] XXXC[SEQ ID NO: 76] CXXX [SEQ ID NO: 77] HCXXX [SEQ ID NO: 78] XXXCH[SEQ ID NO: 79] HCXXC [SEQ ID NO: 80] HCXXS [SEQ ID NO: 81] HCXXT[SEQ ID NO: 82] CXXCH [SEQ ID NO: 83] SXXCH [SEQ ID NO: 84] TXXCH[SEQ ID NO: 85] HCXXCH [SEQ ID NO: 86] XXXXLX [SEQ ID NO: 87] DXXLL[SEQ ID NO: 88] YXXX [SEQ ID NO: 89] HX(0,2)CXX[CST]:  H CXX[CST][SEQ ID NO: 78] HX CXX[CST] [SEQ ID NO: 90] HXXCXX[CST] [SEQ ID NO: 91][CST]xxC(0,2)H:  [CST]XXCH [SEQ ID NO: 79] [CST]XxCXH [SEQ ID NO: 92][CST]XxCXXH [SEQ ID NO: 93] CXXCX(0,2)H:  CXXC H [SEQ ID NO: 83] CXXCX H[SEQ ID NO: 94] CXXCXXH [SEQ ID NO: 95] H(0,2)CXXC:  H CXXC[SEQ ID NO: 83] H XCXXC [SEQ ID NO: 96] HXXCXXC [SEQ ID NO: 97]HX(0,2)XCXXS:  H CXXS [SEQ ID NO: 81] H XCXXS [SEQ ID NO: 98] HXXCXXS[SEQ ID NO: 99] HX(0,2)XCXXT:  H CXXT [SEQ ID NO: 82] H XCXXT[SEQ ID NO: 100] HXXCXXT [SEQ ID NO: 101] SXXCX(0,2)H SXXC H[SEQ ID NO: 84] SXXCX H [SEQ ID NO: 102] SXXCXXH [SEQ ID NO: 103]TXXCX(0,2)H:  TXXC H [SEQ ID NO: 85] TXXCX H [SEQ ID NO: 104] TXXCXXH[SEQ ID NO: 105] HCHXC [SEQ ID NO: 106] CXXHCH [SEQ ID NO: 107]H  CPYCVRSLQPLALEGSLQKRG [SEQ ID NO: 108] HAACPYCVRSLQPLALEGSLQKRG[SEQ ID NO: 109] AHACPYCVRSLQPLALEGSLQKRG [SEQ ID NO: 110]AAACPYCVRSLQPLALEGSLQKRG [SEQ ID NO: 111] AAACHPCVRSLQPLALEGSLQKRG[SEQ ID NO: 112] AAHCHPCVRSLQPLALEGSLQKRG [SEQ ID NO: 113]AAACHGCVRSLQPLALEGSLQKRG [SEQ ID NO: 114]  HXCPYCSRVVHLYRNGKD[SEQ ID NO: 115] HXXCPYCSRVVHLYRNGKD [SEQ ID NO: 116]

1. An isolated immunogenic peptide of between 13 and 100 amino acidscomprising a MHC class II T cell epitope of an antigen, and immediatelyadjacent or separated by at most 7 amino acids from said epitope aH-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78], [SEQ ID NO:90] or [SEQ ID NO:91])or a [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79], [SEQ ID NO:92] or [SEQ IDNO:93]) redox motif sequence for use as a medicament.
 2. The peptide foruse according to claim 1, with the proviso that said antigen does notcontain in its sequence said motif within a distance of 10 amino acidsof said epitope.
 3. The peptide for use according to claim 1, with theproviso that said antigen does not contain in its sequence said motif.4. The peptide for use according to claim 1, wherein the motif isH-X-C-X(2)-[CST] [SEQ ID NO:90] or [CST]-X(2)-C-X-H [SEQ ID NO:92] redoxmotif sequence.
 5. The peptide for use according to claim 1, wherein themotif is H-C-X(2)-[CST] [SEQ ID NO:78] or [CST]-X(2)-C-H [SEQ ID NO:79]redox motif sequence.
 6. The peptide for use according to claim 1,wherein the motif is H-X(0,2)-C-X(2)-C([SEQ ID NO:80], [SEQ ID NO:96] or[SEQ ID NO:97]), or C-X(2)-C-X(0,2)-H ([SEQ ID NO:83], [SEQ ID NO:94] or[SEQ ID NO:95]).
 7. The peptide for use according to claim 1, whereinthe motif is H-C-X(2)-C [SEQ ID NO:80] or C-X(2)-C-H [SEQ ID NO:83]. 8.The peptide for use according to claim 1, wherein said peptide has alength of between 13 and 75 amino acids.
 9. The peptide for useaccording to claim 1, wherein said peptide has a length of between 13and 50 amino acids.
 10. The peptide for use according to claim 1,wherein said peptide has a length of between 13 and 30 amino acids. 11.The peptide for use according to claim 1, wherein the MHC class II Tcell epitope, is separated from said motif by a sequence of at most 4amino acids.
 12. The peptide for use according to claim 1, wherein theMHC class II T cell epitope, is separated from said motif by sequence of2 amino acids.
 13. The peptide for use according to claim 1, wherein Xwithin the redox motif is Gly or Pro.
 14. The peptide for use accordingto claim 1, wherein X within the redox motif is not Cys.
 15. The peptidefor use according to claim 1, wherein X outside the redox motif is notCys, Ser or Thr.
 16. The peptide for use in accordance with claim 1 foruse in the prevention or treatment of multiple sclerosis (MS).
 17. Thepeptide for use according to claim 16, where the antigen is anauto-antigen involved in multiple sclerosis.
 18. The peptide for useaccording to claim 16, wherein the auto-antigen is MOG.
 19. The peptidefor use according to claim 16, wherein the peptide comprises the epitopesequence VVHLYRNGK [SEQ ID NO:3].
 20. The peptide for use according toclaim 16, wherein the peptide has the sequence HCPYCSRVVHLYRNGKD [SEQ IDNO:1], HxCPYCSRVVHLYRNGKD [SEQ ID NO: 115], or HxxCPYCSRVVHLYRNGKD [SEQID NO: 116].
 21. The peptide for use in accordance with claim 1 for usein the prevention or treatment diabetes.
 22. The peptide for use inaccordance with claim 21, wherein the antigen is proinsulin.
 23. Anisolated immunogenic peptide of between 13 and 100 amino acidscomprising a MHC class II T cell epitope of an antigen, and immediatelyadjacent or separated by at most 7 amino acids from said epitope aH-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78] or [SEQ ID NO:90] or [SEQ IDNO:91]) or [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79], [SEQ ID NO:92] or [SEQID NO:93] redox motif sequence, with the proviso that said antigen doesnot contain in its sequence said motif within a distance of 10 aminoacids of said epitope. 24-42. (canceled)
 43. A method for obtaining apopulation CD4+ T cells which are cytolytic against cells antigen, themethod comprising the steps of: providing peripheral blood cells;contacting said cells in vitro with an immunogenic peptide of between 13and 100 amino acids comprising an MHC class II T cell epitope of anantigen, and immediately adjacent or separated by at most 7 amino acidsfrom said epitope a H-X(0,2)-C-X(2)-[CST] ([SEQ ID NO:78], [SEQ IDNO:90] or [SEQ ID NO:91]) or a [CST]-X(2)-C-X(0,2)-H ([SEQ ID NO:79],[SEQ ID NO:92] or [SEQ ID NO:93]) redox motif sequence; and expandingsaid cells in the presence of IL-2.
 44. (canceled)